Exploring the muscle architecture effect on the mechanical behaviour of mouse rotator cuff muscles.

Incorporating detailed muscle architecture aspects into computational models can enable researchers to gain deeper insights into the complexity of muscle function, movement, and performance. In this study, we employed histological, multiphoton image processing, and finite element method techniques to characterise the mechanical dependency on the architectural behaviour of supraspinatus and infraspinatus mouse muscles. While mechanical tests revealed a stiffer passive behaviour in the supraspinatus muscle, the collagen content was found to be two times higher in the infraspinatus. This effect was unveiled by analysing the alignment of fibres during muscle stretch with the 3D models and the parameters obtained in the fitting. Therefore, a strong dependence of muscle behaviour, both active and passive, was found on fibre orientation rather than collagen content.

Delivery of cardiovascular progenitors with biomimetic microcarriers reduces adverse ventricular remodeling in a rat model of chronic myocardial infarction.

Despite tremendous progress in cell-based therapies for heart repair, many challenges still exist. To enhance the therapeutic potential of cell therapy one approach is the combination of cells with biomaterial delivery vehicles. Here, we developed a biomimetic and biodegradable micro-platform based on polymeric microparticles (MPs) capable of maximizing the therapeutic potential of cardiac progenitor cells (CPCs) and explored its efficacy in a rat model of chronic myocardial infarction. The transplantation of CPCs adhered to MPs within the infarcted myocardial microenvironment improved the long-term engraftment of transplanted cells for up to one month. Furthermore, the enhancement of cardiac cellular retention correlated with an increase in functional recovery. In consonance, better tissue remodeling and vasculogenesis were observed in the animals treated with cells attached to MPs, which presented smaller infarct size, thicker right ventricular free wall, fewer deposition of periostin and greater density of vessels than animals treated with CPCs alone. Finally, we were able to show that part of this beneficial effect was mediated by CPC-derived extracellular vesicles (EVs). Taken together, these findings indicate that the biomimetic microcarriers support stem cell survival and increase cardiac function in chronic myocardial infarction through modulation of cardiac remodeling, vasculogenesis and CPCs-EVs mediated therapeutic effects. The biomimetic microcarriers provide a solution for biomaterial-assisted CPC delivery to the heart. STATEMENT OF SIGNIFICANCE: In this study, we evaluate the possibility of using a biomimetic and biodegradable micro-platform to improve cardiovascular progenitor therapy. The strategy reported herein serves as an injectable scaffold for adherent cells due to their excellent injectability through cardiac catheters, capacity for biomimetic three-dimensional stem cell support and controllable biodegradability. In a rat model of chronic myocardial infarction, the biomimetic microcarriers improved cardiac function, reduced chronic cardiac remodeling and increased vasculogenesis through the paracrine signaling of CPCs. We have also shown that extracellular vesicles derived from CPCs cultured on biomimetic substrates display antifibrotic effects, playing an important role in the therapeutic effects of our tissue-engineered approach. Therefore, biomimetic microcarriers represent a promising and effective strategy for biomaterial-assisted CPC delivery to the heart.

NRG1 PLGA MP locally induce macrophage polarisation toward a regenerative phenotype in the heart after acute myocardial infarction.

Neuregulin-1 loaded poly(lactic-co-glycolic acid) (PLGA) microparticles hold great promise for treating acute myocardial infarction, as they have been proved to recover heart function and induce positive heart remodelling in preclinical studies. More recently, the inflammatory response of the heart after acute myocardial infarction (AMI) has been identified as one of the major mechanisms in cardiac tissue remodelling and repair. However, the connection between neuregulin-1 PLGA microparticles and inflammation is still not well characterised. In the present study we assessed this relationship in a mouse AMI model. First, in vitro evidence indicated that neuregulin-1 PLGA microparticles induced a macrophage polarisation toward a regenerative phenotype (CD206+ cells), preventing macrophages from evolving toward the inflammatory phenotype (B7-2+ cells). This correlated with in vivo experiments, where neuregulin-1 PLGA microparticles locally improved the CD206+/B7-2+ ratio. Moreover, neuregulin-1 PLGA microparticles were administered at different time points (15 min, 24, 72 and 168 h) after infarction induction without causing secondary inflammatory issues. The time of treatment administration did not alter the inflammatory response. Taken together, these results suggest that neuregulin-1 PLGA microparticles can be administered depending on the therapeutic window of the encapsulated drug and that they enhance the heart's reparative inflammatory response after acute myocardial infarction, helping cardiac tissue repair.

Biodegradation and heart retention of polymeric microparticles in a rat model of myocardial ischemia.

Poly-lactide-co-glycolide (PLGA) microparticles emerged as one of the most promising strategies to achieve site-specific drug delivery. Although these microparticles have been demonstrated to be effective in several wound healing models, their potential in cardiac regeneration has not yet been fully assessed. The present work sought to explore PLGA microparticles as cardiac drug delivery systems. PLGA microparticles were prepared by Total Recirculation One-Machine System (TROMS) after the formation of a multiple emulsion. Microparticles of different size were prepared and characterized to select the most suitable size for intramyocardial administration. Next, the potential of PLGA microparticles for administration in the heart was assessed in a MI rat model. Particle biodegradation over time and myocardial tissue reaction were studied by routine staining and confocal microscopy. Results showed that microparticles with a diameter of 5 µm were the most compatible with intramyocardial administration in terms of injectability through a 29-gauge needle and tissue response. Particles were present in the heart tissue for up to 3 months post-implantation and no particle migration toward other solid organs was observed, demonstrating good myocardial retention. CD68 immunolabeling revealed 31%, 47% and below 4% microparticle uptake by macrophages 1 week, 1 month, and 3 months after injection, respectively (P<0.001). Taken together, these findings support the feasibility of the developed PLGA microparticles as vehicles for delivering growth factors in the infarcted myocardium.

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model.

AIMS/HYPOTHESIS: Cardiotrophin 1 (CT-1) is a recently described cytokine originally isolated from the heart where it has been shown to play an important role in apoptotic protection of cardiomyocytes and heart hypertrophy. Its beneficial properties have also been described in other organs such as liver and neuromuscular tissue. In the present study, we investigated whether CT-1 can confer protection against pro-apoptotic stimuli in pancreatic beta cells, and its role in insulin secretion and diabetes development. METHODS: The effects of CT-1 on apoptosis and function were studied using MIN6B1 cells and freshly isolated murine pancreatic islets. The impact on the development of diabetes was evaluated in Ct1-null (Ct1 (-/-)) mice (the gene Ct1 is also known as Ctf1) using two streptozotocin (STZ)-induced models of diabetes. RESULTS: CT-1 has a protective effect in MIN6B1 cells and murine islets under the pro-apoptotic stimulus of serum deprivation, which correlates with the expression of B cell lymphoma-extra large, or following exposure to a mixture of cytokines. In addition, CT-1 enhances glucose-stimulated insulin secretion in MIN6B1 cells and this was repressed by inhibitors of phospholipase C. Furthermore, Ct1 (-/-) mice were more prone to develop diabetes, and their glucose tolerance test showed impaired plasma glucose clearance which correlated with decreased pancreatic insulin secretion. CONCLUSIONS/INTERPRETATION: The results obtained from both in vitro and in vivo experiments show that CT-1 improves beta cell function and survival, and protects mice against STZ-induced diabetes.

Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia.

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.

Development and validation of ultra high performance liquid chromatography-mass spectrometry method for LBH589 in mouse plasma and tissues.

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC™ BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/z 349.42→157.95 and of tobramycin at 468.2→163. Calibration curves for the UHPLC method (0.0025-1 µg/mL for plasma and tissue homogenates, equivalent to 0.0357-14.2857 µg/g for tissue samples) showed a linear range of detector responses (r>0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from -2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 µg/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 µg/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice.

Función, volúmenes y masa ventricular izquierda por resonancia magnética en estudios realizados en un modelo animal con secuencias SSFP y FLASH: comparación de los resultados / Left ventricular function, volumes, and mass in MRI studies using SFPP versus FLASH sequences in an animal model

Objetivo Comparar la exactitud de 2 secuencias cine eco de gradiente para cuantificar función, volúmenes y masa ventricular izquierda en un modelo animal. Material y métodosSe estudiaron 10 cerdos miniatura tipo Goettingen (7 machos, 3 hembras; peso medio: 49,8±10,65kg; rango: 35¿65kg) en un equipo 1,5 Tesla con secuencias precesión libre en estado estacionario (SSFP) y eco de gradiente convencionales (FLASH) en respiración libre. Se utilizaron imágenes de 8mm de grosor obtenidas en el eje corto para cuantificar los parámetros del ventrículo izquierdo. Se calcularon la fracción de eyección (FE), volúmenes (volumen telediastólico [VTD], volumen telesistólico [VTS], volumen latido [VL]), masa ventricular y la relación señal ruido (RSR) con cada secuencia. Se estudiaron la correlación y concordancia entre las distintas secuencias para cada variable. ResultadosUtilizando la secuencia SSFP, la FE media estimada fue 77,35±3,13%; VTD medio 61,55±8,64ml; VTS medio 13,83±1,92ml; VL medio 47,72±7,78ml y la masa miocárdica media calculada fue de 75,87±11,44g. Mediante la secuencia FLASH se calculó una FE media de 81,87±2,22%; VTD medio 55,4±8,08ml; VTS medio 10,03±1,87ml; VL medio 45,38±6,83ml, y masa miocárdica media 87,74±15,21g. Se demostró una correlación excelente entre SSFP y FLASH para cuantificar VTD, VL y masa miocárdica (r>0,8) y moderada para VTS y FE (r>0,4). La RSR que se obtuvo con la secuencia SSFP fue significativamente superior a la estimada con la secuencia FLASH (diferencia media 120,94±42,94). ConclusionesCon la secuencia SSFP se obtienen volúmenes ventriculares ligeramente superiores y masa ventricular izquierda ligeramente inferior a los calculados con la secuencia FLASH probablemente por su mayor RSR (AU)

ObjectiveTo compare the accuracy of two cine-gradient-echo sequences to quantify left ventricular function, volumes, and mass in an animal model. Material and methodsWe studied ten Gottingen miniature pigs (seven male, three female; mean weight 49.8±10.65kg; range: 35¿65kg) with a 1.5 Tesla MRI scanner using free-breathing SSFP and FLASH sequences. We used 8-mm short-axis images to estimate left ventricular ejection fraction (EF), volumes (end-diastolic (EDV), end-systolic (ESV), and stroke volume (SV)), mass, and signal-to-noise ratio (SNR) on SSFP and FLASH sequences. We analyzed the correlation and concordance of the two sequences for each variable. ResultsUsing the SSFP sequence, the mean estimated EF was 77.35±3.13%; mean EDV 61.55±8.64ml; mean ESV 13.83±1.92ml; mean SV 47.72±7.78ml; and mean myocardial mass 75.87±11.44g. Using the FLASH sequence, the mean EF was 81.87±2.22%; mean EDV 55.4±8.08ml; mean ESV 10.03±1.87ml; mean SV 45.38±6.83ml; and mean myocardial mass 87.74±15.21g. The correlation between SSFP and FLASH to quantify EDV, SV, and myocardial mass was excellent (r>0.8) and moderate (r>0.4) for quantifying ESV and EF. The SNR in the SSFP sequence was significantly higher than in the FLASH sequence (mean difference 120.94±42.94). ConclusionsIn the SSFP sequence, ventricular volumes are slightly higher and ventricular mass is slightly lower than in the FLASH sequence, probably because of the higher SNR on SSFP sequences (AU)

[Left ventricular function, volumes, and mass in MRI studies using SFPP versus FLASH sequences in an animal model.]. / Función, volúmenes y masa ventricular izquierda por resonancia magnética en estudios realizados en un modelo animal con secuencias SSFP y FLASH: comparación de los resultados.

OBJECTIVE: To compare the accuracy of two cine-gradient-echo sequences to quantify left ventricular function, volumes, and mass in an animal model. MATERIAL AND METHODS: We studied ten Gottingen miniature pigs (seven male, three female; mean weight 49.8+/-10.65kg; range: 35-65kg) with a 1.5 Tesla MRI scanner using free-breathing SSFP and FLASH sequences. We used 8-mm short-axis images to estimate left ventricular ejection fraction (EF), volumes (end-diastolic (EDV), end-systolic (ESV), and stroke volume (SV)), mass, and signal-to-noise ratio (SNR) on SSFP and FLASH sequences. We analyzed the correlation and concordance of the two sequences for each variable. RESULTS: Using the SSFP sequence, the mean estimated EF was 77.35+/-3.13%; mean EDV 61.55+/-8.64ml; mean ESV 13.83+/-1.92ml; mean SV 47.72+/-7.78ml; and mean myocardial mass 75.87+/-11.44g. Using the FLASH sequence, the mean EF was 81.87+/-2.22%; mean EDV 55.4+/-8.08ml; mean ESV 10.03+/-1.87ml; mean SV 45.38+/-6.83ml; and mean myocardial mass 87.74+/-15.21g. The correlation between SSFP and FLASH to quantify EDV, SV, and myocardial mass was excellent (r>0.8) and moderate (r>0.4) for quantifying ESV and EF. The SNR in the SSFP sequence was significantly higher than in the FLASH sequence (mean difference 120.94+/-42.94). CONCLUSIONS: In the SSFP sequence, ventricular volumes are slightly higher and ventricular mass is slightly lower than in the FLASH sequence, probably because of the higher SNR on SSFP sequences.

18F-FDG metabolism in a rat model of chronic infarction: a 17-sector semiquantitative analysis.

UNLABELLED: Strategies to establish the functional benefit of cell therapy in cardiac regeneration and the potential mechanism are needed. AIMS: Development of a semi-quantitative method for non invasive assessment of cardiac viability and function in a rat model of myocardial infarction (MI) based on the use of microPET. ANIMALS, METHODS: Ten rats were subjected to myocardial imaging 2, 7, 14, 30, 60 and 90 days after left coronary artery ligation. Intravenous 18F-fluoro-2-deoxy-2-D-glucose (18F-FDG) was administered and regional 18F activity concentrations per unit area were measured in 17 regions of interest (ROIs) drawn on cardiac polar maps. By comparing the differences in 18F uptake between baseline and each of the follow up time points, parametric polar maps of statistical significance (PPMSS) were calculated. Left ventricular ejection fraction (LVEF) was blindly assessed echocardiographically. All animals were sacrificed for histopathological analysis after 90 days. RESULTS: The diagnostic quality of 18F-FDG microPET images was excellent. PPMSS demonstrated a statistically significant decrease in 18F concentrations as early as 48 hours after MI in 4 of the 17 ROIs (segments 7, 13, 16 and 17; p < 0.05) that persisted throughout the study. Semiquantitative analysis of 18F-FDG uptake correlated with echocardiographic decrease in LVEF (p < 0.001). CONCLUSION: The use of PPMSS based on 18F-FDG-microPET provides valuable semi-quantitative information of heart glucose metabolism allowing for non-invasive follow up thus representing a useful strategy for assessment of novel therapies in cardiac regeneration.

Regeneración miocárdica: el futuro al alcance de la mano / Myocardial regeneration: future at reach

En los últimos años hemos asistido a un interés creciente por eltratamiento de la insuficiencia cardíaca mediante el trasplante decélulas madre. Mientras que los estudios con células madre de músculo(mioblastos) se iniciaron hace mas de 10 años, la posibilidadde que las células madre de la médula ósea tengan un enorme potencialde diferenciación y proliferación han estimulado la investigacióncon otros tipos de células madre. Estos estudios experimentales handemostrado, en no pocas ocasiones, resultados contradictorios loque ha llevado a posturas enfrentadas en cuanto a la ética de iniciarestudios clínicos. Creemos que es adecuado tratar de ofrecer unavisión crítica sobre la utilización de las células madre en la insuficienciacardíaca. Quizá la pregunta mas difícil de contestar en estemomento es, si la realización de ensayos clínicos esta justificado ono a la luz de los conocimientos actuales o si por el contrario debemosadquirir un conocimiento mucho más preciso de la posibleeficacia de este tipo de tratamiento y de los mecanismos que justificandicha eficacia, antes de siquiera iniciar los estudios en humanos.En nuestra opinión existen suficientes evidencias que justifican eldesarrollo de ensayos clínicos a pesar de que, sin duda, existenmuchos interrogantes que debemos resolver mediante estudios experimentalesen animales

The last few years have witnessed a growing interest in regenerative therapy of the failing heart by cell transplantation. Initial studies with skeletal myoblasts were conducted more than 10 years ago. However, the potential of bone marrow derived cells has more recently led to a flurry of experimental studies generating overall positive but occasionally conflicting results. The ethics of initiating clinical trials with stem cells in patients with heart failure has been questioned. Although laboratory research attempts to overcome a number of questions surrounding the usefulness and safety of cell therapy, the accumulated body of evidence warrants implementation of clinical trials. The earliest of these have now documented the feasibility of cell therapy. It is now appropriate to conduct safety and efficacy studies which, if carefully done, should allow assessment of the extent to which this concept of regenerative therapy can be made a clinical reality (AU)

[Myocardial regeneration: future at reach]. / Regeneración miocárdica: el futuro al alcance de la mano.

The last few years have witnessed a growing interest in regenerative therapy of the failing heart by cell transplantation. Initial studies with skeletal myoblasts were conducted more than 10 years ago. However, the potential of bone marrow derived cells has more recently led to a flurry of experimental studies generating overall positive but occasionally conflicting results. The ethics of initiating clinical trials with stem cells in patients with heart failure has been questioned. Although laboratory research attempts to overcome a number of questions surrounding the usefulness and safety of cell therapy, the accumulated body of evidence warrants implementation of clinical trials. The earliest of these have now documented the feasibility of cell therapy. It is now appropriate to conduct safety and efficacy studies which, if carefully done, should allow assessment of the extent to which this concept of regenerative therapy can be made a clinical reality.