Multi-modal assessment of a cardiac stem cell therapy reveals distinct modulation of regional scar properties.

BACKGROUND: The initial idea of functional tissue replacement has shifted to the concept that injected cells positively modulate myocardial healing by a non-specific immune response of the transplanted cells within the target tissue. This alleged local modification of the scar requires assessment of regional properties of the left ventricular wall in addition to commonly applied measures of global morphological and functional parameters. Hence, we aimed at investigating the effect of cardiac cell therapy with cardiovascular progenitor cells, so-called cardiac induced cells, on both global and regional properties of the left ventricle by a multimodal imaging approach in a mouse model. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending artery, the therapy group received an intramyocardial injection of 1 × 106 cardiac induced cells suspended in matrigel, the control group received matrigel only. [18F]FDG positron emission tomography imaging was performed after 17 days, to assess regional glucose metabolism. Three weeks after myocardial infarction, cardiac magnetic resonance imaging was performed for morphological and functional assessment of the left ventricle. Following these measurements, hearts were excised for histological examinations. RESULTS: Cell therapy had no significant effect on global morphological parameters. Similarly, there was no difference in scar size and capillary density between therapy and control group. However, there was a significant improvement in contractile function of the left ventricle - left ventricular ejection fraction, stroke volume and cardiac output. Regional analysis of the left ventricle identified changes of wall properties in the scar area as the putative mechanism. Cell therapy reduced the thinning of the scar and significantly improved its radial contractility. Furthermore, the metabolic defect, assessed by [18F]FDG, was significantly reduced by the cell therapy. CONCLUSION: Our data support the relevance of extending the assessment of global left ventricular parameters by a structured regional wall analysis for the evaluation of therapies targeting at modulation of healing myocardium. This approach will enable a deeper understanding of mechanisms underlying the effect of experimental regenerative therapies, thus paving the way for a successful translation into clinical application.

Cardiac cell therapies for the treatment of acute myocardial infarction in mice: systematic review and meta-analysis.

Backgound Aims: This meta-analysis aims at summarizing the whole body of research on cell therapies for acute myocardial infarction (MI) in the mouse model to bring forward ongoing research in this field of regenerative medicine. Despite rather modest effects in clinical trials, pre-clinical studies continue to report beneficial effects of cardiac cell therapies for cardiac repair following acute ischemic injury. Results: The authors' meta-analysis of data from 166 mouse studies comprising 257 experimental groups demonstrated a significant improvement in left ventricular ejection fraction of 10.21% after cell therapy compared with control animals. Subgroup analysis indicated that second-generation cell therapies such as cardiac progenitor cells and pluripotent stem cell derivatives had the highest therapeutic potential for minimizing myocardial damage post-MI. Conclusions: Whereas the vision of functional tissue replacement has been replaced by the concept of regional scar modulation in most of the investigated studies, rather basic methods for assessing cardiac function were most frequently used. Hence, future studies will highly benefit from integrating methods for assessment of regional wall properties to evolve a deeper understanding of how to modulate cardiac healing after acute MI.

Expedient assessment of post-infarct remodeling by native cardiac magnetic resonance imaging in mice.

Novel therapeutic strategies aiming at improving the healing process after an acute myocardial infarction are currently under intense investigation. The mouse model plays a central role for deciphering the underlying mechanisms on a molecular and cellular level. Therefore, we intended to assess in-vivo post-infarct remodeling as comprehensively as possible using an expedient native magnetic resonance imaging (MRI) in the two most prominent infarct models, permanent ligation (PL) of the left anterior descending artery (LAD) versus ischemia reperfusion (I/R). Mice were subjected to either permanent or transient (45 min) occlusion of the LAD. After 3 weeks, examinations were performed with a 7-Tesla small animal MRI system. Data analysis was performed with the freely available software Segment. PL resulted in a massive dilation of the left ventricle, accompanied by hypertrophy of the non-infarcted myocardium and a decline of contractile function. These effects were less pronounced following I/R compared to healthy animals. Single plane assessments were not sufficient to capture the specific differences of left ventricular (LV) properties between the two infarct models. Bulls-eye plots were found to be an ideal tool for qualitative LV wall assessment, whereas a multi-slice sector-based analysis of wall regions is ideal to determine differences in hypertrophy, lateral wall thinning and wall thickening on a quantitative level. We combine the use of polar map-based analysis of LV wall properties with volumetric measurements using simple CINE CMR imaging. Our strategy represents a versatile and easily available tool for serial assessment of the LV during the remodeling process. Our study contributes to a better understanding of the effects of novel therapies targeting the healing of damaged myocardium.

[68Ga]-NODAGA-RGD Positron Emission Tomography (PET) for Assessment of Post Myocardial Infarction Angiogenesis as a Predictor for Left Ventricular Remodeling in Mice after Cardiac Stem Cell Therapy.

Angiogenesis plays a central role in the healing process following acute myocardial infarction. The PET tracer [68Ga]-NODAGA-RGD, which is a ligand for the αvß3 integrin, has been investigated for imaging angiogenesis in the process of healing myocardium in both animal and clinical studies. It´s value as a prognostic marker of functional outcome remains unclear. Therefore, the aim of this work was to establish [68Ga]-NODAGA-RGD for imaging angiogenesis in the murine infarct model and evaluate the tracer as a predictor for cardiac remodeling in the context of cardiac stem cell therapy. [68Ga]-NODAGA-RGD PET performed seven days after left anterior descending coronary artery (LAD) occlusion in 129S6 mice showed intense tracer accumulation within the infarct region. The specificity was shown in a sub-group of animals by application of the competitive inhibitor cilengitide prior to tracer injection in a subgroup of animals. Myocardial infarction (MI) significantly reduced cardiac function and resulted in pronounced left ventricular remodeling after three weeks, as measured by cardiac MRI in a separate group. Cardiac induced cells (CiC) that were derived from mESC injected intramyocardially in the therapy group significantly improved left ventricular ejection fraction (LVEF). Surprisingly, CiC transplantation resulted in significantly lower tracer accumulation seven days after MI induction. Accordingly, we successfully established the PET tracer [68Ga]-NODAGA-RGD for the assessment of αvß3 integrin expression in the healing process after MI in the mouse model. Yet, our results indicate that the mere extent of angiogenesis following MI does not serve as a sufficient prognostic marker for functional outcome.

18F-FDG PET-Based Imaging of Myocardial Inflammation Following Acute Myocardial Infarction in a Mouse Model.

Cellular inflammation is an integral part of the healing process following acute myocardial infarction and has been under intense investigation for both therapeutic and prognostic approaches. Monocytes and macrophages are metabolically highly active and show increased uptake rates of glucose and its analog, 18F-FDG. Yet, the specific allocation of the radioactivity to the inflammatory cells via positron emission tomography (PET) imaging requires the suppression of glucose metabolism in viable myocardium. In mice, the most important model organism in basic research, this can be achieved by the application of ketamine/xylazine (KX) for anesthesia instead of isoflurane. Yet, while the consensus exists that glucose metabolism is effectively suppressed, a strategy for reproducible image analysis is grossly lacking and causes uncertainty concerning data interpretation. We introduce a simple strategy for systematic image analysis, which is a prerequisite to evaluate therapies targeting myocardial inflammation. Mice underwent permanent occlusion of the left anterior descending artery (LAD), inducing an acute myocardial infarction (MI). Five days after MI induction, 10MBq 18F-FDG was injected intravenously and a static PET/CT scan under ketamine/xylazine anesthesia was performed. For image reconstruction, we used an algorithm based on three-dimensional ordered subsets expectation maximization (3D-OSEM) followed by three-dimensional ordinary Poisson maximum a priori (MAP) reconstruction. Using this approach, high focal tracer uptake was typically located in the border zone of the infarct by visual inspection. To precisely demarcate the border zone for reproducible volume of interest (VOI) positioning, our protocol relies on positioning VOIs around the whole left ventricle, the inferobasal wall and the anterolateral wall guided by anatomical landmarks. This strategy enables comparable data in mouse studies, which is an important prerequisite for using a PET-based assessment of myocardial inflammation as a prognostic tool in therapeutic applications.

Spray- and laser-assisted biomaterial processing for fast and efficient autologous cell-plus-matrix tissue engineering.

At present, intensive investigation aims at the creation of optimal valvular prostheses. We introduced and tested the applicability and functionality of two advanced cell-plus-matrix seeding technologies, spray-assisted bioprocessing (SaBP) and laser-assisted bioprocessing (LaBP), for autologous tissue engineering (TE) of bioresorbable artificial grafts. For SaBP, human mesenchymal stem cells (HMSCs), umbilical cord vein endothelial cells (HUVECs) and fibrin were simultaneously spray-administered on poly(ε-caprolactone) (PCL) substrates. For LaBP, HUVECs and HMSCs were separately laser-printed in stripes, followed by fibrin sealing. Three-leaflet valves were manufactured following TE of electrospun PCL tissue equivalents. Grafts were monitored in vitro under static and dynamic conditions in bioreactors. SaBP and LaBP resulted in TE of grafts with homogeneous cell distribution and accurate cell pattern, respectively. The engineered valves demonstrated immediate sufficient performance, complete cell coating, proliferation, engraftment, HUVEC-mediated invasion, HMSC differentiation and extracellular matrix deposition. SaBP revealed higher efficiency, with at least 12-fold shorter processing time than the applied LaBP set-up. LaBP realized coating with higher cell density and minimal cell-scaffold distance. Fibrin and PCL stability remain issues for improvement. The introduced TE technologies resulted in complete valvular cell-plus-matrix coating, excellent engraftment and HMSCs differentiation. SaBP might have potential for intraoperative table-side TE considering the procedural duration and ease of implementation. LaBP might accelerate engraftment with precise patterns.

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice.

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.

Autologous valve replacement-CD133+ stem cell-plus-fibrin composite-based sprayed cell seeding for intraoperative heart valve tissue engineering.

OBJECTIVE: The development of biological valve prostheses with lifetime native-like performance and optimal host engraftment is an ultimate goal of heart valve tissue engineering. We describe a new concept for autologous graft coating based on a CD133(+)-stem-cells-plus-fibrin (SC+F) complex processed from bone marrow and peripheral blood of a single patient. METHODS: CD133(+)-SC (1 × 10(6) cells/mL) from human bone marrow and autologous fibrin (20 mg/mL) were administered simultaneously via spray administration using the novel Vivostat Co-Delivery System. During static cultivation, SC+F performance was monitored for 20 days after delivery and compared with controls. For dynamic testing SC+F-composite was sprayed on a decellularized porcine pulmonary valve and transferred to a bioreactor under pulsatile flow conditions for 7 days. RESULTS: Static cultivation of SC+F-composite induced significant improvements in stem cell proliferation as compared with controls. For dynamic testing, microscopic analyses on a smooth engineered heart valve surface detected homogenous distribution of stem cells. Ultrasonic analysis revealed native-like valve performance. Applied CD133(+) stem cells differentiated into endothelial-like cells positive for CD31 and vascular endothelial growth factor receptor 2 and engrafted the valve. However, occasional delamination was observed. CONCLUSION: SC+F serves as an excellent autologous matrix for intraoperative tissue engineering of valve prostheses promising optimal in vivo integration. However, stability remains an issue.

Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Matrigel promotes angiogenesis in the myocardium from ischemic injury and prevents remodelling of the left ventricle. We assessed the therapeutic efficacy of intracardiac matrigel injection and matrigel-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, matrigel (250 µl) or phosphate-buffered solution (PBS) was delivered by intracardiac injection. Compared to the MI control group (MI-PBS), matrigel significantly improved left ventricular function (n= 11, P < 0.05) assessed by pressure-volume loops after 4 weeks. There is no significant difference in infarct size between MI-matrigel (MI-M; 21.48 ± 1.49%, n = 10) and MI-PBS hearts (20.98 ± 1.25%, n = 10). The infarct wall thickness of left ventricle is significantly higher (P < 0.01) in MI-M (0.72 ± 0.02 mm, n = 10) compared with MI-PBS (0.62 ± 0.02 mm, n = 10). MI-M hearts exhibited higher capillary density (border 130.8 ± 4.7 versus 115.4 ± 6.0, P < 0.05; vessels per high-power field [HPF; 400×], n = 6) than MI-PBS hearts. c-Kit(+) stem cells (38.3 ± 5.3 versus 25.7 ± 1.5 c-Kit(+) cells per HPF [630×], n = 5, P < 0.05) and CD34(+) cells (13.0 ± 1.51 versus 5.6 ± 0.68 CD34(+) cells per HPF [630×], n = 5, P < 0.01) were significantly more numerous in MI-M than in MI-PBS in the infarcted hearts (n = 5, P < 0.05). Intracardiac matrigel injection restores myocardial functions following MI, which may attribute to the improved recruitment of CD34(+) and c-Kit(+) stem cells.

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n =11-14, P < 0.05) and decreased right ventricular wall stress (n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 +/- 0.03% versus 0.42 +/- 0.03%; n = 6, P= 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit(+) and CD34(+) stem cells via the enhanced expression of chemoattractant SDF-1.

Localized and sustained SDF-1 gene release mediated by fibronectin films: A potential method for recruiting stem cells.

Gene-activated matrix has wide potential utilization in tissue engineering. It may genetically modify cells with plasmid DNA encoding therapeutic genes and allow sustained expression and release of the proteins to surrounding tissues. In this study, we assessed the feasibility of the local gene release from human fibronectin (HFN) substrate and the efficacy of local release of stromal cell-derived factor-1 (SDF-1) gene on c-kit+ cell homing. Cationic polymer polyethylenimine (25kDa PEI) was used as non-viral DNA vector. Gene-activated HFN (GAH) was prepared by mixing PEI/DNA complexes with HFN substrate. The DNA retardation, the complex size, and the DNA release speed from the GAH were studied. The in vitro transfection was optimized by luciferase expression and cell viability assay in the COS7 cell line. Localized gene expression in COS7 cells cultured on the GAH was assessed by LacZ and GFP-N3-SDF-1 marker genes. Ckit+ cell homing was investigated in response to the local in vitro SDF-1 expression from rat mesenchymal stem cells (RMSCs) cultured on GAH. Results showed GAH allows long time-sustained DNA release, localized gene delivery, and high transfection efficiency. Local SDF-1 expression with GAH is a promising method to induce targetable stem cell homing.

Single high-dose intramyocardial administration of erythropoietin promotes early intracardiac proliferation, proves safety and restores cardiac performance after myocardial infarction in rats.

Various studies demonstrate erythropoietin (EPO) as a cardioprotective growth hormone. Recent findings reveal EPO in addition might induce proliferation cascades inside myocardium. We aimed to evaluate whether a single high-dose intramyocardial EPO administration safely elevates early intracardiac cell proliferation after myocardial infarction (MI). Following permanent MI in rats EPO (3000 U/kg) in MI EPO-treatment group (n=99) or saline in MI control group (n=95) was injected along the infarction border. Intramyocardial EPO injection activated the genes of cyclin D1 and cell division cycle 2 kinase (cdc2) at 24 h after MI (n=6, P<0.05) evaluated by real time-PCR. The number of Ki-67+ intracardiac cells analyzed following immunohistochemistry was significantly enhanced by 45% in the peri-infarction zone at 48 h after EPO treatment (n=6, P<0.001). Capillary density was significantly enhanced by 17% as early as seven days (n=6, P<0.001). After six weeks, left ventricular performance assessed by conductance catheters was restored under baseline and dobutamine induced stress conditions (n=11-14, P<0.05). No thrombus formation was observed in the heart and in distant organs. No deleterious systemic adverse effects were apparent. Single high-dose intramyocardial EPO delivery proved safety and promoted early intracardiac cell proliferation, which might in part have contributed to an attenuated myocardial functional decline.