ABSTRACT
Myocardial infarction (MI) is one of the leading causes of death in the world. With the improvement of clinical therapy, the mortality of acute MI has been significantly reduced. However, as for the long-term impact of MI on cardiac remodeling and cardiac function, there is no effective prevention and treatment measures. Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has anti-apoptotic and pro-angiogenetic effects. Studies have shown that EPO plays a protective role in cardiomyocytes in cardiovascular diseases, such as cardiac ischemia injury and heart failure. EPO has been demonstrated to protect ischemic myocardium and improve MI repair by promoting the activation of cardiac progenitor cells (CPCs). This study aimed to investigate whether EPO can promote MI repair by enhancing the activity of stem cell antigen 1 positive stem cells (Sca-1+ SCs). Darbepoetin alpha (a long-acting EPO analog, EPOanlg) was injected into the border zone of MI in adult mice. Infarct size, cardiac remodeling and performance, cardiomyocyte apoptosis and microvessel density were measured. Lin- Sca-1+ SCs were isolated from neonatal and adult mouse hearts by magnetic sorting technology, and were used to identify the colony forming ability and the effect of EPO, respectively. The results showed that, compared to MI alone, EPOanlg reduced the infarct percentage, cardiomyocyte apoptosis ratio and left ventricular (LV) chamber dilatation, improved cardiac performance, and increased the numbers of coronary microvessels in vivo. In vitro, EPO increased the proliferation, migration and clone formation of Lin- Sca-1+ SCs likely via the EPO receptor and downstream STAT-5/p38 MAPK signaling pathways. These results suggest that EPO participates in the repair process of MI by activating Sca-1+ SCs.
Subject(s)
Animals , Mice , Ventricular Remodeling , Erythropoietin , Myocardial Infarction , Heart , Stem CellsABSTRACT
Cardiac stem cells (CSCs) transplantation has been recognized to be effective on the treatment of myocardial infarction (MI), but some techniques still need to be developed in the isolation and culture of CSCs, which is the key problem restricting the clinical application of CSCs. This study was focused on the isolation of Lin(lineage-negative) Sca-1(stem cell antigen-1-positive) CSCs from newborn C57BL/6J mice (0-3 d) by mixed enzymatic-explant isolation in combination with immunomagnetic separation. The digesting time, digesting frequency, incubation temperature, stirring speed, centrifugation time and rotational speed were strictly controlled in the experiment. In order to increase the survival rate of CSCs, the medium changing time and manner were optimized in primary CSCs culture. The percentages of Sca-1cells in primary and passage cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) the proportion of LinSca-1cells within the collected cells could be as high as (85.03 ± 5.60)% after isolation and purification; (2) In vitro culture of LinSca-1CSCs grew into spheres on the 5day, and over the whole bottom of the dish on the 7day. The growth curve showed that the cells were in logarithmic growth phase on the 3day; (3) Immunofluorescence staining data showed that the expression of Sca-1, the CSCs membrane-specific marker, was decreased after subculture, and flow cytometry data showed that the percentages of Sca-1cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)% in passage 1 (P1), P3, and P5 CSCs, respectively. The above results suggest that high purity of LinSca-1CSCs can be obtained by enzymolysis combined with immunomagnetic separation method. Moreover, the CSCs culture system is stable. In our experiment, the Sca-1CSCs isolation and culture method has been successfully established, and it is simple, stable, effective and reliable. The method can provide a stable methodological basis for the treatment of MI by LinSca-1CSCs transplantation.
ABSTRACT
Since the autoantibodies against the second extracellular loop of β(1)-adrenoceptor (β(1)-AABs) have been found in the sera of patients with idiopathic dilated cardiomyopathy (IDCM), the involvement of autoimmune mechanisms in the pathogenesis of many cardiovascular diseases has extensively been investigated. Our previous study found that urinary occult blood and protein excretion were frequently found in the rats with positive β(1)-AABs, but the mechanisms are unclear. Therefore, we infused the β(1)-AABs into the vein periodically in an attempt to investigate whether β(1)-AABs could induce morphological and functional changes in the kidneys of adult and aged rats and explore the possible mechanisms. The synthetic peptide according to the sequences of the second extracellular loop of β(1)-adrenoceptor (β(1)-AR-ECII) was used to immunize the adult rats to acquire enough β(1)-AABs for use. Neonatal rat ventricular myocytes (NRVMs) culture was used to observe the biological effects of β(1)-AABs on the beating rate. The purified β(1)-AABs were transfused into the vein of rats. The sera level of blood urea nitrogen (BUN), creatinine (CR), uric acid (UA), urinary specific gravity, protein excretion, occult blood and urinary glucose were detected at the different time points by biochemistry and urine analyzers. HE and Masson's trichrome staining were used to detect the changes in kidney structure of passively immunized rats. Enhanced green fluorescent protein (EGFP) and β(1)-AR-EGFP plasmids were transfected into the human embryonic kidney 293 (HEK293) cells in order to observe the changes in cell injury with the treatment of β(1)-AABs. It was found that the sera level of BUN, CR and UA increased gradually and the ratio of BUN to CR decreased progressively with the administration of β(1)-AABs. The increasing of proteinuria, urinary occult blood and urinary glucose was detected by urine analyzer in β(1)-AABs group. By HE and Masson's coloration, lots of mononuclear cell infiltration and collagen fibers deposition could be observed at the 24th week of immunization. After the treatment of β(1)-AABs, the caspase-3 activity increased significantly in the HEK293 cells transfected with β(1)-AR-EGFP plasmids, while no significant changes were observed for lactate dehydrogenase (LDH) activity. The results indicate that long-term presence of β(1)-AABs can induce the morphological and functional damage of the kidneys in adult and aged rats.