Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization of hematopoietic cells. We previously found that FL-mobilized hematopoietic ...stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit(+) Sca-1(+)Lin(-)(KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kit(hi) and c-Kit(lo) Sca-1(+)Lin(-) cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations.
c-Kit(hi) or c-Kit(lo) KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kit(hi) CD34(-)Flt3(-) KSL, c-Kit CD34(+)Flt3(-) KSL, c-Kit(hi) CD34(+)Flt3(+) KSL were investigated in an in vivo transplantation model.
Only FL-mobilized PB c-Kit(hi) KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kit(hi) CD34(+)Flt3(-) cells (short-term HSC) and c-Kit(hi) CD34(-)Flt3(-) KSL cells (long-term HSC). There was a significant decrease in c-Kit CD34Flt3 KSL late multipotent progenitors in PB. A combination of c-Kit(hi) CD34Flt3 and c-Kit CD34(+)Flt3(-) KSL cells offered the most effective rescue of ablated recipients.
These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.
Administration of Cu/Zn superoxide dismutase (SOD) without catalase fails to alleviate myocardial stunning, but extracellular SOD (Ec-SOD) may be more effective because it binds to heparan sulfate ...proteoglycans on the cellular glycocalyx. We therefore used in vivo gene transfer to increase systemic levels of Ec-SOD and determined whether this gene therapy protects against myocardial stunning.
The cDNA for human Ec-SOD was cloned behind the cytomegalovirus (CMV) promoter and incorporated into a replication-deficient adenovirus (Ad5/CMV/Ec-SOD). Injection of this virus (2x10(8) pfu/kg IV) produced high levels of Ec-SOD in the liver, which could be redistributed to the heart and other organs by injection of heparin. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days starting 3 days after intravenous injection of Ad5/CMV/Ec-SOD or Ad5/CMV/nls/LacZ (negative control). Both groups were given heparin (2000 U/kg IV) 2 hours before the first sequence of occlusions. The severity of myocardial stunning was measured as the total deficit of LV wall thickening after the last reperfusion. On day 1, the total deficit of wall thickening was markedly decreased in Ad5/CMV/Ec-SOD rabbits versus controls and similar to that seen on days 2 and 3 in controls.
The results demonstrate that in vivo gene transfer of the cDNA encoding Ec-SOD provides the heart with substantial protection against myocardial stunning without the need for concomitant administration of catalase. The present observations provide the basis for controlling gene therapy at the posttranslational level and for simultaneously protecting multiple organs from oxidant stress.
The objective of this study was to use electrocardiogram (ECG)-independent parameters to determine whether preconditioning (PC) exists in humans during percutaneous transluminal coronary angioplasty ...(PTCA).
Several studies suggest that both ischemia and adenosine induce PC in the human heart during PTCA. However, because almost all of these studies relied on ST-segment shifts as indicators of the severity of ischemia, their conclusions continue to be questioned, and the very existence of ischemic or adenosine PC in humans remains controversial.
Eighteen patients received either intracoronary adenosine (n = 9) or normal saline (n = 9); 10 min later, they underwent PTCA (three 2-min balloon inflations 5 min apart).
Compared with the first inflation, in untreated patients the second and third inflations were associated with less systolic dysfunction (two-dimensional echocardiography), less diastolic dysfunction (color M-mode echocardiography), less lactate production, and less H+release into the great cardiac venous blood. In adenosine-treated patients, the extent of all of these abnormalities during the first inflation was less than in untreated patients and did not change with subsequent inflations.
Previous exposure to a brief episode of ischemia (first balloon inflation) or to adenosine produces concordant decreases in ECG, subjective, mechanical, and metabolic manifestations of ischemia during PTCA. These data support the concept that both ischemic PC and pharmacologic PC exist in humans and that PTCA is a useful clinical setting in which to discern their mechanism.
Abstract only
The objective of this study was to determine whether asymmetric division of human cardiac progenitor cells (hCPCs) occurs by:
random DNA template segregation;
selective retention of the ...old template DNA strand; or
a combination of both processes.
Myocardial samples were enzymatically dissociated and hCPCs were sorted for the stem cell antigen c-kit. During in vitro expansion, hCPCs were exposed to BrdU for 36 hours to reach a 90% degree of labeling. BrdU-tagged hCPCs were plated at limiting dilution to obtain single cell-derived clones. Sixty clones comprising 10 –125 c-kit-positive hCPCs developed in 7–10 days. In four cases, one single BrdU-bright hCPC was identified while the remaining clonogenic cells were negative for the halogenated nucleotide. In these clones, the number of BrdU-negative hCPCs was 25, 85, 95, and 111. Conversely, in 56 clones hCPCs were uniformly labeled and showed very low levels of BrdU. In a second group of experiments, hCPCs in late-anaphase initial-telophase were identified and the distribution of BrdU in the two clusters of chromosomes was analyzed. In 5% of mitotic cells, three-dimensional reconstruction by confocal microscopy documented that BrdU-labeling was restricted at one pole only of the dividing hCPCs. PCNA which is highly expressed in newly synthesized DNA was restricted to the BrdU-negative chromosomes. In a third set of studies, hCPCs were loaded with quantum dots, cultured for 36 hours in the presence of BrdU and examined 96 hours later. Quantum dots are progressively diluted by cell division independently from the modality of DNA template segregation. Thus, hCPCs with minimal levels of quantum dots and bright BrdU localization were interpreted as replicating cells which retained the old DNA strand. By this approach, 5% hCPCs displayed these two critical properties. The uneven distribution of the cell fate determinants Numb and α-adaptin confirmed that hCPCs underwent asymmetric division. In conclusion, these data support the hypothesis that immortal DNA strand cosegregation participates in asymmetric kinetics of hCPCs although random-segregation of DNA template is the prevailing mechanism of hCPC growth.
Studies with the JAK‐STAT inhibitor AG‐490 suggest that activation of STAT1 and STAT3 is necessary for the delayed protection afforded by ischemic preconditioning (PC). However, the individual role ...of STAT1 in late PC‐induced cardioprotection remains unclear. We used STAT1−/− mice to investigate the role of STAT1 in mediating the protective effects of ischemic PC and the expression of cardioprotective proteins (iNOS, COX‐2, and HO‐1).
In wild‐type mice, ischemic PC induced phosphorylation of STAT1 (pTyr(701) and pSer(727)) and increased STAT‐DNA binding activity 30 min later; 24 h later, PC increased expression of iNOS, COX‐2, and HO‐1, and reduced infarct size. The expression and phosphorylation of STAT1 was eliminated in STAT1−/− mice. Deletion of STAT1 did not affect the expression or the phosphorylation of STAT3. STAT1 deletion inhibited the PC‐induced increase in STAT‐DNA binding activity, blocked the PC‐induced upregulation of iNOS, COX‐2, and HO‐1, and attenuated the protection of PC against myocardial infarction.
We conclude that STAT1 activates a pro‐survival mechanism that protects against cardiac cell death during late PC. In contrast to previous in vitro studies, our results in vivo demonstrate that STAT1 plays an obligatory role in PC‐induced upregulation of cardioprotective proteins and in the delayed cardioprotection afforded by ischemic PC.
National AHA 0575035N and NIH R01 HL‐65660 and P01 HL‐78825
Repair of the damaged heart Leri, Annarosa; Kajstura, Jan; Quaini, Federico ...
Kidney international,
November 2005, 2005-11-00, 20051101, Letnik:
68, Številka:
5
Journal Article
Recenzirano
Odprti dostop
The human heart contains a subpopulation of replicating myocytes that is enhanced in pathologic states characterized by acute and chronic cardiac failure. In accordance with our hypothesis that these ...dividing cells represent amplifying myocytes originated from a pool of undifferentiated cells, effort was made to establish the conditions for the isolation and expansion of potential progenitor cells from small samples of human myocardium. Here we report the identification of a c-kit-positive cardiac stem cell that is self-renewing, clonogenic, and multipotent. This primitive cell has the morphologic properties and expresses some of the surface antigens commonly found in mesenchymal stem cells. Additionally, when locally injected in the infarcted myocardium of immunodeficient rats and mice, cardiac stem cells regenerate myocytes and coronary vessels.
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