These studies demonstrate, for the first time, that BMCs expressing the c-kit are preferably retained in the heart following ischemia-reperfusion injury. This selectivity varies in time following the ischemic insult with a delay in the onset of selectivity and greatest selectivity seen after 30 min of reperfusion. The optimum engraftment time of 30 min reperfusion also corresponds with the post-ischemia time required for type 1 activation of the endothelium, during which P-selectin molecules surface from intracellular Weibel-Palade bodies [11
]. In contrast, type II activation, which requires transcription, generally requires hours [12
] and is likely not responsible for the results we report. Our results are also in excellent temporal agreement with in vivo P-selectin-dependent increases in overall leukocyte rolling following activation of cremaster endothelium conducted by Ley, et. al. [13
]. P-selectin is further implicated as the mediating mechanism for this selective retention by our antibody neutralization experiments, which found that P-selectin, but not L-selectin, was required to produce selective engraftment of c-kit+
BMCs and by histological demonstration that engrafted c-kit+
cells are usually found adjacent to cells expressing P-selectin. Our analysis of the BMCs further demonstrated that c-kit+
BMCs are generally positive for PSGL-1, the primary P-selectin ligand in leukocytes, while only about half of c-kit−
BMCs express this adhesion molecule. Together, these results indicate that P-selectin/PSGL-1 mediate selective engraftment of c-kit+
cells to the heart early after ischemia-reperfusion stress.
Complementing the ex-vivo studies, in vitro exploration of P- and L-selectins, which have been shown to play a part in the inflammatory cell adhesion process [14
], showed a strong selective interaction of c-kit+
BMCs with P-selectin. While c-kit+
cells demonstrated a statistically-significant decrease in rolling velocity along with a 9-fold increase in firm adhesion from L- to P-selectin, c-kit− BMCs showed nearly identical dynamics regardless of the selectin substrate. As P-selectin is not highly expressed on resting endothelium but is known to surface when the endothelium is activated [15
], the enhanced P-selectin interaction for c-kit+
BMCs supports the mechanistic hypothesis that ischemia-induced activation of the coronary endothelium results in P-selectin-mediated selective retention of c-kit+
Although the in vivo regenerative capacity of c-kit+
BMCs remains controversial, these cells have been shown to differentiate into cardiomyocytes in vivo [17
]. Additional angiogenic paracrine signaling effects have been identified for c-kit+
BMCs in the injured heart [19
]. Thus, the strong retention for these cells in response to injury (selectivity ratio of 18±2) may indicate initiation of a repair response. Together, these findings provide new insights into the dynamics of cell recruitment to the injured heart
while highlighting the utility and versatility of the IPMH model for mechanistic studies focusing on endogenous repair and translational studies aimed at developing or refining new therapeutic strategies.
The features of preferential engraftment of c-kit+
BMCs to the heart following ischemia-reperfusion stress in these studies provides clues to the mechanisms of this dynamic process. Most importantly, the preferential recruitment in an IPMH model obviates a requirement for systemic hemodynamic, neural or biochemical triggers for preferential engraftment. However, an auxillary role for such extracardiac factors is not excluded by these studies. Additional factors may be causally related to the selective retention we have described. For example, because ischemia durations of 10 and 30 min. release a burst of reactive oxygen species (ROS) in isolated-perfused rabbit hearts upon reperfusion, the 15 min. ischemia duration utilized in these studies is expected to produce a significant ROS burst that could activate the coronary endothelium [20
]. These factors may be causally related to cell engraftment and should be further elucidated to complete our understanding of the mechanistic pathway.
Despite the versatility of the IPMH model for studies of cell engraftment, a few limitations deserve mention. For example, we directly infused filtered, labeled bone marrow constituents into the IPMH while the in vivo setting involves an indirect interaction in which marrow-derived cells are released into the circulation and the circulation, in turn, perfuses the myocardium. While our procedure may have allowed some marrow-derived populations greater access of to the heart than occurs in vivo, this realization does not diminish the validity of injury-triggered engraftment cues and preferential engraftment dynamics revealed by these studies. Moreover, the utility of this model is likely greatest for studying early engraftment dynamics before deterioration of contractile performance and edema of the IPMH model reduce its reproducibility and relevance to in vivo dynamics. Additionally, this model is critically dependent on rapid cannulation of the mouse heart since delay will induce ischemic insults that would confound the interpretation of protocol-driven ischemia-reperfusion stress. To address this concern, we routinely abort experiments if the heart cannulation and initiation of perfusion takes more than two minutes.
Myocardial ischemia-reperfusion stress provokes time-dependent and highly selective engraftment of c-kit+ cells included in the perfusate via a P-selectin-dependent mechanism. In this context, additional applications of this model might include more extensive examinations how the type of ischemia (demand vs. supply), duration of ischemia or alternative local cytokine manipulations might alter engraftment dynamics. More detailed profiling of selectively retained cells via further immunophenotyping, sorting with gene expression studies, and/or characterization of the IPMH effluent should provide additional insights into the precise sequence of events responsible for the highly selective engraftment dynamics we observed. Refined mechanistic hypotheses raised by such studies could be testing by gain- and loss-of-function studies enabled by altering the myocardial cytokine milieu, exploiting genetically-manipulated mouse models and/or the varying the composition of the infused cells. For example, manipulations of the infused cell populations via sorting and/recombination could permit unique opportunities to examine whether interactions among infused cells affect engraftment dynamics.
- Cardiac Ischemia-Reperfusion Injury Results in Selective Engraftment of c-kit+ BMCs
- c-kit+ Selectivity Begins at 30 Min of Reperfusion and Persists Beyond 60 Min
- c-kit+ Selectivity is Dependent on P-selectin but not L-selectin