Mesenchymal Stem Cells Pretreatment With Stromal-Derived Factor-1 Alpha Augments Cardiac Function and Angiogenesis in Infarcted Myocardium.

BACKGROUND: Stem cell therapy is among the novel approaches for the treatment of post-myocardial infarction cardiomyopathy. This study aims to compare the effect of stromal-derived factor 1 α (SDF1α), mesenchymal stem cells (MSCs) in combination with the lentiviral production of vascular endothelial growth factor (VEGF) on infarct area, vascularization and eventually cardiac function in a rat model of myocardial infarction (MI). METHODS: The influence of SDf1α on MSCs survival was investigated. MSCs were transduced via a lentiviral vector containing VEGF. After that, the effect of mesenchymal stem cell transfection of VEGF-A165 and SDf1α preconditioning on cardiac function and scar size was investigated in five groups of MI rat models. The MSC survival, cardiac function, scar size, angiogenesis, and lymphocyte count were assessed 72 hours and 6 weeks after cell transplantation. RESULTS: SDF1α decreased the lactate dehydrogenase release in MSCs significantly. Also, the number of viable cells in the SDF1α-pretreated group was meaningfully more than the control. The left ventricular systolic function significantly enhanced in groups with p240MSC, SDF1αMSC, and VEGF-A165MSC in comparison to the control group. CONCLUSIONS: These findings suggest that SDF1α pretreatment and overexpressing VEGF in MSCs could augment the MSCs' survival in the infarcted myocardium, reduce the scar size, and improve the cardiac systolic function.

The greater effect of high-intensity interval training versus moderate-intensity continuous training on cardioprotection against ischemia-reperfusion injury through Klotho levels and attenuate of myocardial TRPC6 expression.

BACKGROUND: Myocardial ischemia-reperfusion (IR) injury is a leading cause of death all over the world, so developing practical approaches to promote cardioprotection against IR injury is essential. Exercise training is an effective strategy to improve cardioprotection. Hence, the purpose of this study was to investigate the effect of short-term preconditioning with two types of high-intensity interval training (HIIT) and moderate intensity continuous training (MICT) on klotho and TRPC6 mechanisms in cardioprotection. METHODS: Eighty Male Wistar rats (250-300 g) were randomly divided into 7 groups, including Control, HIIT, MICT, Sham, IR, HIIT+IR, and MICT+IR. Training was performed in 5 consecutive days. HIIT protocol consisted of running on the treadmill at intervals 85-90% vo2max that separated by slow intensity periods at 50-60% vo2max. MICT program was performed at 70% VO2max at the same running distance with HIIT groups. The cardiac IR injury was induced by LAD occlusion followed by reperfusion. ELISA kit was used in order to measure the plasma levels of klotho, LDH and CK-MB, and TRPC6 expression was determined using the western blot technique. Data were analyzed using one way ANOVA and Tukey's post hoc tests. RESULTS: The results of this study showed that both types of exercise training programs significantly increase plasma levels of klotho and reduce the infarct size and heart injury. In addition, the exercise training decreased the amount of TRPC6 channels expression during IR. However, the effect of HIIT on increasing the klotho and cardioprotection was greater compared to MICT. CONCLUSIONS: Based on the results, even a short-term of aerobic exercise training, especially HIIT, promotes cardioprotection against IR injury and decreases infarct size via an increase in klotho and attenuate of protein expression of myocardial TRPC6 during IR.

Mesenchymal stem cells from human amniotic membrane differentiate into cardiomyocytes and endothelial-like cells without improving cardiac function after surgical administration in rat model of chronic heart failure.

Introduction: Human amnion-derived mesenchymal stem cells (hAMSCs) have been used in the treatment of acute myocardial infarction. In the current study, we investigated the efficacy of hAMSCs for the treatment of chronic model of myocardial ischemia and heart failure (HF) in rats. Methods: Male Wistar rats weighing between 250 to 350 g were randomized into three groups: sham, HF control and HF+hAMSCs. For HF induction, animals were anesthetized and underwent left anterior descending artery ligation. In HF+hAMSCs group, 2×106 cells were injected into the left ventricular muscle four weeks post ischemia in the border zone of the ischemic area. Cardiac function was studied using echocardiography. Masson's trichrome staining was used for studying tissue fibrosis. Cells were transduced with green fluorescent protein (GFP) coding lentiviral vector. Immunohistochemistry was used for detecting GFP, vascular-endothelial growth factor (VEGF) and troponin T markers in the tissue sections. Results: Assessment of the cardiac function revealed no improvement in the myocardial function compared to the control HF group. Moreover, tissue fibrosis was similar in two groups. Immunohistochemical study revealed the homing of the injected hAMSCs to the myocardium. Cells were stained positive for VEGF and troponin T markers. Conclusion: injection of hAMSCs 4 weeks after ischemia does not improve cardiac function and cardiac muscle fibrosis, although the cells show markers of differentiation into vascular endothelial cells and cardiomyocytes. In sum, it appears that hAMSCs are effective in the early phases of myocardial ischemia and does not offer a significant advantage in patients with chronic HF.

Gdn 3+@CNTs-PEG versus Gadovist®: In Vitro Assay.

OBJECTIVES: Carbon nanotubes (CNTs) are allotropes of carbon with a length-to-diameter ratio greater than 106 with the potential uses as medical diagnostic or therapeutic agents. In vitro studies have revealed that gadolinium (Gd) nanoparticle-catalyzed single-walled carbon nanotubes (SWCNTs) possess superparamagnetic properties, which enable them to be used as contrast agents in magnetic resonance imaging (MRI). Our study synthesized Gd-CNT for use as MRI contrast agents. METHODS: To reduce the toxicity and solubility of CNTs, it was functionalized, and after loading with Gd was coated with polyethylene glycols (PEG). We then synthesized different concentrations of Gdn 3+@CNTs-PEG and Gadovist® to be evaluated as MRI contrast agents. RESULTS: The analysis showed that the Gd concentration in Gadovist® was 12.18% higher than synthesized Gdn 3+@CNTs-PEG, but the mean signal intensity of the Gdn 3+@CNTs-PEG was approximately 3.3% times higher than Gadovist®. CONCLUSIONS: Our findings indicate that synthesized Gdn 3+@CNTs-PEG has the potential to be used as an MRI contrast agent in vitro, but in vivo assessment is necessary to determine the bio-distribution, kinetic, and signal enhancement characteristics.

High-intensity interval training increase GATA4, CITED4 and c-Kit and decreases C/EBPß in rats after myocardial infarction.

AIM: Myocardial infarction (MI), an important cause of morbidity and mortality, can be followed by left ventricular dysfunction and cardiomyocyte loss. Cardiac repair mechanisms may subsequently improve left ventricular function. Exercise training has been suggested to have cardioprotective effects against MI damage, but detailed knowledge is lacking on the effects of different types and intensities of exercise training on molecular targets of cardiomyocyte regeneration. MAIN METHODS: MI was induced in male Wistar rats by ligating the left anterior descending coronary artery. After MI induction, the rats were randomly assigned to one of five groups: sham operated, and experimental MI followed by no exercise, or low, moderate or high intensity exercise Cardiac function and infarct size were assessed by echocardiography and Evans blue/TTC staining, respectively. The expression of mRNA markers and proteins associated with myocardial regeneration was measured with RT-PCR and western blotting. KEY FINDINGS: Exercise training at different intensities improved cardiac function and levels of stem cell and cardiomyocyte markers, and reduced infarct size. mRNA levels of GATA4, Nkx2.5 and c-Kit and protein expression of Nkx2.5 and c-Kit were significantly increased in all MI-exercise groups. The high-intensity exercise group had greater increases than the low and moderate intensity exercise groups. In the high-intensity exercise group, Sca-1 and CITED4 increased more than in the low-intensity exercise group. C/EBPß mRNA and protein levels decreased after exercise training, with greater reductions in the high-intensity exercise group than the low- or moderate-intensity groups. SIGNIFICANCE: The findings suggest that by targeting cardiogenesis, high-intensity training can exert cardioprotective effects against cardiac dysfunction in an experimental model of MI.

Targeting necroptotic cell death pathway by high-intensity interval training (HIIT) decreases development of post-ischemic adverse remodelling after myocardial ischemia / reperfusion injury.

Regulated necrosis (necroptosis) plays a pivotal role in the extent of cardiomyocyte loss and the development of post-ischemic adverse remodelling and cardiac dysfunction following myocardial I/R injury. Although HIIT has been reported to give rise to cardioprotection against MI, but the detailed knowledge of its molecular targets for treatment of MI is still not available. The LAD of Male Wistar rats was occluded to induce MI for 30 min and reperfusion for eight weeks. We investigated the effect of long-term HIIT for eight weeks on lipid peroxidation, SOD activity and GSH content using ELISA assay. Cardiac function, fibrosis, and infarct size were assessed by echocardiography, Masson's trichrome and Evans Blue/TTC dual staining respectively. The expressions of gene markers of myocardial hypertrophy, fibrosis and key mediators of necroptosis were measured using RT-PCR and western blotting assay respectively. The results indicated that HIIT reduced lipid peroxidation, infarct size and improved endogenous antioxidant system and heart function. Significant decreases in mRNA levels of procollagen α1(I), α1(III), and fibronectin1were observed following HIIT. Moreover, that HIIT significantly decreased the expression of key mediators of necroptosis induced by MI (P < 0.05). There were no significant differences in ß-MHC mRNA level in different groups. The findings of study suggest that HIIT might exert cardioprotective effects against post-ischemic adverse remodeling through targeting necroptosis process. Likewise, cardioprotective effects of HIIT in coping with myocardial I/R injury may be associated with RIP1-RIP3-MLKL axis. These findings establish a critical foundation for higher efficiency of exercise-based cardiac rehabilitation post-MI and future research.

Transient Activation of Reprogramming Transcription Factors Using Protein Transduction Facilitates Conversion of Human Fibroblasts Toward Cardiomyocyte-Like Cells.

Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine). The cells showed cardiac phenotype over a period of 3 weeks without first undergoing reprogramming into or through a pluripotent intermediate, shown by lack of expression of key pluripotency markers. p-iCLCs exhibited cardiac features at both the gene and protein levels. Our study provides an alternative method for the generation of p-iCLCs which shortcut reprogramming toward allogeneic cardiomyocytes in a safe and efficient manner and could facilitate generation of genetic material-free cardiomyocytes.

Improvement of Heart Failure by Human Amniotic Mesenchymal Stromal Cell Transplantation in Rats.

Background: Recently, stem cells have been considered for the treatment of heart diseases, but no marked improvement has been recorded. This is the first study to examine the functional and histological effects of the transplantation of human amniotic mesenchymal stromal cells (hAMSCs) in rats with heart failure (HF). Methods: This study was conducted in the years 2014 and 2015. 35 male Wistar rats were randomly assigned into 5 equal experimental groups (7 rats each) as 1- Control 2- Heart Failure (HF) 3- Sham 4- Culture media 5- Stem Cell Transplantation (SCT). Heart failure was induced using 170 mg/kg/d of isoproterenol subcutaneously injection in 4 consecutive days. The failure confirmed by the rat cardiac echocardiography on day 28. In SCT group, 3×106 cells in 150 µl of culture media were transplanted to the myocardium. At the end, echocardiographic and hemodynamic parameters together with histological evaluation were done. Results: Echocardiography results showed that cardiac ejection fraction in HF group increased from 58/73 ± 9% to 81/25 ± 6/05% in SCT group (p value < 0.001). Fraction shortening in HF group was increased from 27/53 ± 8/58% into 45/55 ± 6/91% in SCT group (p value < 0.001). Furthermore, hAMSCs therapy significantly improved mean diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, rate pressure product, and left ventricular end-diastolic pressure compared to those in the HF group, with the values reaching the normal levels in the control group. A marked reduction in fibrosis tissue was also found in the SCT group (p value < 0.001) compared with the animals in the HF group. Conclusion: The transplantation of hAMSCs in rats with heart failure not only decreased the level of fibrosis but also conferred significant improvement in heart performance in terms of echocardiographic and hemodynamic parameters.

Echocardiographic evaluation of cardiac function in ischemic rats: value of m-mode echocardiography.

BACKGROUND: Echocardiography is a well-established diagnostic tool for a safe, reproducible and accurate evaluation of cardiac anatomy, hemodynamics and function in clinical practice. OBJECTIVES: We sought to demonstrate the efficacy and feasibility of M-mode echocardiography to evaluate cardiac structure and function in normal and MI-induced adult rats. MATERIALS AND METHODS: All animal procedures were approved by the ethics committee of Tehran University of Medical Sciences and the investigation conformed to the "Guide for the Care and Use of Laboratory Animals" published by the United States National Institutes of Health. Forty-eight male Wistar rats weighing 280-300 grams were obtained from a single breeding colony. The statistical analyses were performed using SPSS 20.0. RESULTS: Echocardiographic measurements were possible in all rats before and after the operation. In our survey, we studied echocardiographic alterations in rats after MI induction. Changes can be seen in all echocardiographic mean values after myocardial infarction (MI), but significant decrease (P < 0.01) of Fractional shortening and Ejection Fraction as well as significant increase (P < 0.05) of end systolic diameter and systolic volume after left anterior descending coronary artery (LAD) ligation can be good signs of MI induction. CONCLUSIONS: In light of our results, it can be concluded that we succeeded in establishing a precise echocardiographic method to confidently assess the success of LAD ligation surgery in rats. It is feasible to thoroughly monitor the functional efficiency of regional therapeutic interventions such as intra-myocardial stem cell injection.

Neurogenesis and increase in differentiated neural cell survival via phosphorylation of Akt1 after fluoxetine treatment of stem cells.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI). Its action is possibly through an increase in neural cell survival. The mechanism of improved survival rate of neurons by FLX may relate to the overexpression of some kinases such as Akt protein. Akt1 (a serine/threonine kinase) plays a key role in the modulation of cell proliferation and survival. Our study evaluated the effects of FLX on mesenchymal stem cell (MSC) fate and Akt1 phosphorylation levels in MSCs. Evaluation tests included reverse transcriptase polymerase chain reaction, western blot, and immunocytochemistry assays. Nestin, MAP-2, and ß-tubulin were detected after neurogenesis as neural markers. Ten µ M of FLX upregulated phosphorylation of Akt1 protein in induced hEnSC significantly. Also FLX did increase viability of these MSCs. Continuous FLX treatment after neurogenesis elevated the survival rate of differentiated neural cells probably by enhanced induction of Akt1 phosphorylation. This study addresses a novel role of FLX in neurogenesis and differentiated neural cell survival that may contribute to explaining the therapeutic action of fluoxetine in regenerative pharmacology.

Outcome improvement of cellular cardiomyoplasty using triple therapy: mesenchymal stem cell+erythropoietin+vascular endothelial growth factor.

To improve cellular cardiomyoplasty efficacy after myocardial infarction (MI), we postulated that combining mesenchymal stem cells (MSCs) transplantation with anti-apoptotic and angiogenic effects of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) may provide better prognosis in an infarcted heart 48 rats, underwent left anterior descending artery ligation, were divided into eight groups and treated as follows: Group 1: MSC+EPO+VEGF, Group 2: MSC+EPO, Group 3: MSC+VEGF, Group 4: MSC, Group 5: EPO+VEGF, Group 6: EPO, Group 7: VEGF and Group 8: Control. After MI induction, EPO and VEGF were injected subcutaneously at the dose of 3000 U/kg and 3 µg/kg respectively. MSCs were transplanted one week after MI. In the fourteenth and sixteenth days after infarction, EPO was injected again. Echocardiography demonstrated that all treatments improved left ventricular function significantly (before vs. after treatment) but in control group ejection fraction deteriorated over the 2-months period. Percent of ejection fraction recovery in all treatment groups were significantly greater than control (P<0.05). Compared with the control group, all treatments attenuated cell death in peri-infarct areas significantly, except groups 6 and 7. Vascular density of all treatment groups were more than control group but this superiority was statistically significant only in group 1 (P<0.01). All of our treatments had beneficial effects to some extent but MSC transplantation combined with EPO and VEGF administration resulted in superior therapeutic outcome in enhancing cell survival and neovascularization.

Dehydroepiandrosterone stimulates nerve growth factor and brain derived neurotrophic factor in cortical neurons.

Due to the increasing cases of neurodegenerative diseases in recent years, the eventual goal of nerve repair is very important. One approach for achieving a neuronal cell induction is by regenerative pharmacology. Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are neurotrophins that play roles in neuronal development, differentiation, and protection. On the other hand, dehydroepiandrosterone (DHEA) is a neurosteroid which has multiple actions in the nervous system. DHEA could be an important agent in regenerative pharmacology for neuronal differentiation during tissue regeneration. In this study, we investigated the possible role of DHEA to modulate NGF and BDNF production. The in vivo level of neurotrophins expression was demonstrated by ELISA in rat harvested brain cortex. Also neurotrophins expression after DHEA treatment was revealed by the increased neurite extension, immunostaining, and BrdU labeling in rats. Anti-NGF and anti-BDNF antibodies were used as suppressive agents on neurogenesis. The results showed that NGF and BDNF are overproduced after DHEA treatment but there is not any overexpression for NT-3 and NT-4. Also DHEA increased neurite extension and neural cell proliferation significantly. Overall, DHEA might induce NGF and BDNF neurotrophins overproduction in cortical neurons which promotes neural cell protection, survival, and proliferation.