Endothelial Progenitor Cells Are Rapidly Recruited to Myocardium and Mediate Protective Effect of Ischemic Preconditioning via "Imported" Ni
http://www.100md.com
循环学杂志 2005年第3期
the Division of Cardiovascular Research (M.I., A.I., A.W., E.E., D.W.L.), Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Mass
Regenerative Medicine (H.N., T.A.), Institute of Biomedical Research and Innovation, Kobe, Japan.
Abstract
Background— The function of bone marrow–derived endothelial progenitor cells (EPCs) in repair of ischemic tissue has been the subject of intense scrutiny, and the capacity of these cells to contribute significantly to new blood vessels remains controversial. The possibility that EPCs could act as reservoirs of cytokines has been implied by several observations; however, a specific role for cytokine delivery has not been identified.
Methods and Results— We performed a series of experiments that revealed the rapid recruitment of EPCs to the myocardium by very short periods of ischemia, so-called ischemic preconditioning. The recruited EPCs express an array of potentially cardioprotective cytokines including nitric oxide synthase isoforms. Bone marrow transplantation studies, using donor marrow null for nitric oxide synthase isoforms, revealed that both endothelial and inducible nitric oxide synthase derived from bone marrow cells play essential roles in the cardioprotective effect that normally occurs after ischemic preconditioning.
Conclusions— These findings provide novel insights about the role of bone marrow–derived cells in ischemic preconditioning and also reveal that distinct mechanisms regulate recovery after ischemia-reperfusion and chronic ischemic injury.
Key Words: myocardial infarction ; ischemia ; nitric oxide synthase ; blood cells
Introduction
The notion that repetitive transient sublethal interruption of the blood supply to the myocardium, resulting in ischemia, followed by restoration of the blood supply, or reperfusion (ischemia-reperfusion [I-R]), can have a protective effect against subsequent prolonged ischemia has been established by clinical observation and experimental studies over the past 2 decades.1–4 The myocardial protection that results from ischemic preconditioning (IP) is characterized by reduced infarct size, decreased incidence of fatal arrhythmias, and reduced postischemic contractile cardiac dysfunction.4–6 Multiple mechanisms have been suggested to play a role in IP,7–10 including vascular endothelial growth factor (VEGF)–mediated neovascularization during myocardial ischemia11–13 protein kinase C activation,2,4,13–16 and nitric oxide synthase (NOS) activity,17–19 among others.9,20
Angiogenesis has also been shown to play a central role in the recovery of the myocardium after ischemia and infarction.12,13 We and others have identified endothelial progenitor cells (EPCs) in adult human peripheral blood21,22 and have shown that EPCs accumulate in active angiogenic foci and participate in neovascularization after ischemic insults, a concept consistent with postnatal vasculogenesis.23–25 Moreover, studies have shown that augmentation of the supply of bone marrow–derived progenitor cells improves outcome after ischemic injury.26–28 Nevertheless, the actual contribution of EPC-derived vessel formation to improved outcome has been controversial,29 and there has been a suggestion that the extent of physiological benefit that results from augmentation of the EPC supply in sites of ischemia has been greater than the apparent anatomic contribution of these cells in the newly formed vasculature.
Accordingly, we performed a series of investigations to test the hypothesis that EPCs could exert a protective effect in the setting of myocardial ischemia via the rapid delivery of cytokines. We used the model of IP because this simple maneuver has been shown to have dramatic protective effects on myocardial preservation and has been associated with the expression of a number of potentially protective cytokines. Initial pilot studies revealed a strikingly rapid recruitment of EPCs after transient IP and relatively minor EPC-mediated neovascularization compared with the degree of cardioprotection.
Methods
Mice and Bone Marrow Transplantation Model
Male FVB/NJ or C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me) aged 4 to 6 weeks were used as recipients for bone marrow transplantation (BMT). Transgenic mice of FVB/N-TgN (Tie-2-LacZ) 182Sato, eNOS–/– mice (B6.129P2-Nos3tmlUnc), or iNOS–/– mice (B6.129P2-Nos2tmlLau; Jackson Laboratories) were used as donors for the BMT. All subsequent procedures were performed on BMT mice. St. Elizabeth’s Institutional Animal Care and Use Committee approved this research protocol, including all procedures and animal care.
The procedure of BMT was performed as described previously.23,24 Briefly, the background/recipient mice were lethally irradiated for bone marrow ablation with 9.0 Gy for FVB/NJ mice and 12.0 Gy for C57BL/6J mice; each received 1 half million donor bone marrow mononuclear cells. At 6 to 8 weeks after BMT, by which time the bone marrow of the recipient mice was reconstituted, all the procedures described below were performed. Hearts of BMT mice were harvested at the indicated time points after surgery for histology.
Surgical Procedure
The mouse model of myocardial ischemia was based on that described previously.5,7 Briefly, after induction of anesthesia and mechanical ventilation, a left thoracotomy was performed in the fourth intercostal space, followed by pericardiectomy. An 8-0 monofilament nylon suture was passed under the left anterior descending coronary artery (LAD) just proximal to the first diagonal branch. A 5-mm section of polyethylene suture was placed on top of the LAD to secure the ligation without damaging the artery. Both ends of the suture were passed through a segment of flared PE10 tubing to form a snare. IP was induced by pulling the snare and clamping tube with 4 cycles of 4 minutes of LAD occlusion and 4 minutes of reperfusion. I-R injury was induced with 45 minutes of LAD occlusion, and chronic ischemic injury was induced with permanent LAD ligation. Control mice underwent thoracotomy and pericardiectomy followed by the passing of a suture under the LAD without interruption of blood flow for 30 minutes as a sham operation for comparison with IP.
Immunohistochemistry
The hearts of BMT mice were harvested at predetermined times after surgery and prepared for frozen tissue sectioning. Double-fluorescent immunohistochemistry was performed with an antibody against ;-galactosidase (;-gal) and endothelial NOS (eNOS), inducible NOS (iNOS), or VEGF. Nonspecific protein binding was blocked with 10% normal horse serum. Sections were incubated with rabbit polyclonal anti-;-gal antibody (1:250, Cortex) at 4°C overnight, followed by Cy3-goat anti-rabbit IgG (1:500, Jackson ImmunoResearch) as a secondary antibody for 30 minutes. Goat polyclonal anti-eNOS, -iNOS and -VEGF antibodies (1:250, Santa Cruz) and Cy2-donky anti-goat IgG (1:500, Jackson ImmunoResearch) were used as a secondary primary and its secondary antibody, respectively. Normal rabbit or goat IgG served as negative controls. The endothelial cell–specific marker FITC-isolectin-B4 (1:100; Vector Laboratories) was used for capillary staining. Nuclei were counterstained with DAPI (1:5000, Sigma), and sections were mounted in aqueous mounting medium. Images were examined with a fluorescent microscope (Nikon ECLIPSE TE200).
EPC Culture Assay and Bone Marrow–Derived EPC Culture
EPC culture assay and bone marrow–derived EPC culture was performed as described previously.24,30 Briefly, mononuclear cells isolated from 500 μL of peripheral blood were cultured in 5% FBS/EBM-2 (Clonetics) medium with supplements (SingleQuot Kit; Clonetics) on rat vitronectin (Sigma) with 0.1% gelatin-coated 4-well glass chamber slides. After 4 days in culture, cells were coincubated with DiI-acLDL (Biomedical Technologies) for 1 hour, followed by FITC-BS-1 lectin staining. The dual-stained cells, considered EPCs, were counted in 10 randomly selected high-power fields under a fluorescent microscope. Bone marrow–derived mononuclear cells of isolated tibia and femur were plated on cell culture dishes coated with rat vitronectin at a density of 5x105/cm2 and cultured in 5% FBS/EBM-2 medium. After 4 days in culture, nonadherent cells were removed, and adherent cells were reseeded at a density of 5x104/cm2. After 3 days in further culture, the cells were used as EPC-rich cell population for Western blot analysis.
Western Blot Analysis
Western blot analysis was performed as described previously.31 Briefly, cells cultured in EBM-2 medium with supplements under certain culture conditions were lysed in sample buffer in a 35-mm culture dish. Protein extracts were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane (BioRad). The membranes were blocked with 10% nonfat dry milk, then immunoblotted overnight at 4°C with rabbit polyclonal antibodies against mouse eNOS (1:1000, BD Pharmingen), phospho-eNOS (1:1000, Cell Signaling), iNOS (1:1000, BD Pharmingen), and VEGF (1:500, Santa Cruz) and a goat polyclonal antibody against -actin (1:1000, Santa Cruz). After 45 minutes in incubation with horseradish peroxidase–goat anti-rabbit IgG (1: 5000, Santa Cruz), immunoreactive bands were visualized with ECL reagent (Amersham). Densitometric analyses for the blots were performed with NIH Image software.
Statistical Analysis
Data are presented as mean±SEM. Significance (P<0.05) was determined by ANOVA followed by post hoc analysis with the Fisher procedure.
Results
EPCs Are Recruited to the Myocardium Immediately After IP Alone
To investigate EPC kinetics in peripheral blood and the myocardium after IP alone, we performed EPC culture assays and histological evaluations in Tie-2/LacZ BMT mice, which receive bone marrow from transgenic mice that constitutively express ;-gal regulated by the promoter of the endothelial gene, Tie-2.32,33 As shown in Figure 1a, the number of circulating EPCs decreased immediately after IP to a significantly greater extent than in a sham control group (n=6 at the indicated time points, respectively; 33.2±3.3 EPCs per high-power field [HPF] in cell culture assay at baseline, 21.5±1.9 EPCs/HPF in sham versus 11.7±1.3 EPCs/HPF in IP at 1 hour, P<0.01). Circulating EPC counts were then similar for the next few hours (sham=8.7±1.4 EPCs/HPF versus IP=8.3±0.8 EPCs/HPF at 3 hours, P=NS; sham=16.0±1.5 EPCs/HPF versus IP=19.8±2.7 EPCs/HPF at 6 hours, P=NS) and then increased significantly in the IP group, peaking at 3 days (21.2±1.7 versus 33.7±3.5 EPCs/HPF at 18 hours, 32.0±3.3 versus 42.7±4.7 EPCs/HPF at 1 day, and 41.5±1.4 versus 59.1±1.6 EPCs/HPF at 3 days; P<0.01). By day 7, the levels in both groups had returned toward baseline levels, although they remained higher in the IP group (29.7±4.2 versus 37.8±2.4 EPCs/HPF, P<0.01). To assess the microvascular architecture, in situ fluorescent staining with the endothelial cell–specific marker FITC-conjugated BS1-lectin (Vector Laboratories) was performed with minor modification as described previously.24 Briefly, 10 minutes after systemic injection of BS1-lectin (0.1 mg per mouse), hearts were fixed with 2% paraformaldehyde for 2 hours and prepared for frozen cross-sectioning. Immunofluorescent staining revealed that the early, precipitous decrease in circulating EPCs in the IP group coincided with rapid EPC recruitment to the jeopardized zone of the myocardium. As shown in Figure 1b, EPCs, identified by red fluorescence resulting from ;-gal immunoreactivity, appeared in the myocardium immediately, 1 and 3 hours after IP, and gradually diminished thereafter.
These studies revealed that repetitive transient LAD occlusion and reperfusion induced dramatic recruitment of EPCs into the ischemic zone of the myocardium immediately after IP alone. This was accompanied by a biphasic modulation of EPC kinetics in the peripheral circulation, with a rapid decrease in circulating EPCs that coincided with the appearance of EPCs in the myocardium. Thus, EPC kinetics in peripheral blood and recruitment of EPCs in the myocardium suggested a dynamic equilibrium. Subsequently, the increased number of EPCs in the late phases after IP may have resulted from EPC mobilization by VEGF (or other cytokines) released by the ischemic myocardium (data not shown). Although a sham operation, including thoracotomy and pericardiectomy, can also affect EPC kinetics, this more modest response is thought to be due to the wound-healing process.23 The implication is that recruitment of bone marrow–derived cells to sites of injury, previously referred to under the rubric of "inflammation," may comprise a variety of responses, with some targeted directly to the mechanisms of tissue repair or vascular preservation. Indeed, most of the recruited cells in the myocardium immediately after IP were not inflammatory cells expressing markers of CD3 and CD13 (data not shown) but were Tie-2–expressing cells, consistent with EPCs.
IP Reduces Infarct Size and Preserves Capillary Density After I-R Injury
Next, we sought to document the extent of myocardial protection by IP, and its relationship to the recruitment of EPCs, by inducing myocardial infarction by prolonged ischemia followed by reperfusion (I-R) with or without preceding IP. Consistent with the pilot studies of IP alone, the number of ;-gal–expressing cells (indicating bone marrow origin and Tie-2 expression) in the peri-infarct area was significantly higher in the IP group than in the sham group (10.6±2.4 versus 43.0±4.9/mm2, P<0.01; Figure 2a). Also as expected, the extent of myocardial damage induced by I-R injury was decreased in the IP group (n=5) compared with the sham group (n=5) at 7 days. IP significantly reduced infarct size (2.4±0.2 mm2 in the sham group versus 1.0±0.2 mm2 in the IP group; 58% reduction, P<0.01; Figure 2b). Representative photographs of immunofluorescent staining with in situ perfusion of BS1-lectin (green) to identify capillaries in the ischemic area are shown in Figure 3a. Capillary density (in 3 random HPFs) in the ischemic area 3 days after I-R injury was significantly greater in the IP group than in the sham group, which suggests that IP had preserved the microvasculature (101.5±16.8/HPF versus 239.0±7.8/HPF, P<0.01; Figure 3b). The participation of EPCs, indicated by double staining of red (;-gal, ie, bone marrow–derived Tie-2–expressing cells) and green (endothelium of any origin), also was observed in capillaries of the myocardium (Figure 3a). The extent of incorporated EPCs in the microvasculature of the ischemic area was also significantly higher in the IP group than in the sham group (25.5±2.1/HPF versus 58.3±5.2/HPF, P<0.01; Figure 3c). This indicates that bone marrow–derived cells had incorporated into the myocardial microvasculature in greater numbers when ischemia-induced injury was preceded by IP.
Incorporated EPCs Produce eNOS, iNOS, and VEGF in Ischemic Myocardium
Double fluorescent immunostaining in ischemic myocardium 3 days after surgery in the IP group revealed that the incorporated ;-gal–expressing EPCs (red) and some of the cardiomyocytes in ischemic myocardium expressed eNOS, iNOS, and VEGF (green; Figure 4a). Merged images reveal large numbers of double-positive cells expressing ;-gal (Tie-2–expressing cells of bone marrow origin) and eNOS, iNOS, and VEGF, respectively, which reveals that the EPCs were significant sources of cardioprotective factors.
To clarify the regulation of EPC-produced cytokines in ischemic myocardium, we next performed an in vitro study using cultured EPCs under hypoxic conditions in an attempt to mimic myocardial ischemia. EPCs isolated from bone marrow were cultured under normoxic (95% air and 5% CO2) or hypoxic (<2% oxygen, 95% N2 and 5% CO2)34 conditions in a standard incubator at 37°C. Cells were then further exposed to either hypoxia or normoxia 24 hours after hypoxia for the indicated time. In Western blot analyses, EPC phospho-eNOS abundance was gradually and slightly reduced by persistent hypoxia (arbitrary units, 1.0 in normoxia versus 0.6±0.10 after 24 hours of hypoxia, P<0.05; Figure 4b), whereas expression of iNOS and, to a lesser extent, VEGF was significantly induced (iNOS, 0 in normoxia versus 0.2±0.02 at 8 hours and 1.3±0.15 at 24 hours in hypoxia, P<0.0001; VEGF, 1.0 in normoxia versus 1.4±0.12 at 8 hours and 1.4±0.17 at 24 hours in hypoxia, P<0.01; Figures 4c and 4d). In contrast, hypoxia-induced repression of eNOS expression was gradually reversed in normoxia (arbitrary units, 1.0 in 24 hours of hypoxia versus 1.5±0.09 after 8 hours and 1.8±0.06 after 24 hours in normoxia, P<0.001; Figure 4b). Similarly, the upregulation of iNOS and VEGF expression induced by hypoxia was significantly reversed by normoxia (iNOS, 1.0 in 24 hours of hypoxia versus 0.6±0.09, 0.3±0.06, and 0 after 4, 8, and 24 hours in normoxia, respectively, P<0.0001; VEGF, 1.0 in 24 hours of hypoxia versus 0.9±0.04 and 0.5±0.04 after 8 and 24 hours in normoxia, P<0.05 and <0.0001, respectively; Figures 4c and 4d). Although these data may not necessarily reflect changes in gene expression by EPCs in the myocardium, they do provide a starting point for our investigation of the potential mechanisms involved.
These findings show that EPC expression of the active form of eNOS, phospho-eNOS, appears to be diminished by hypoxia. Conversely, iNOS in EPCs is induced by hypoxia, as is endogenous VEGF.34 The upregulation of iNOS and VEGF is also reversed by normoxia. Hypoxia promotes EPC migration but inhibits EPC mitogenic activity in response to VEGF (data not shown).34
To integrate these results, EPCs recruited to the ischemic myocardium are stimulated by the local tissue environment and upregulate expression of NOS and VEGF. Sustained tissue hypoxia, which reflects chronic ischemic injury in vivo, not only promotes migration activity of EPCs but also further enhances EPC VEGF production and iNOS expression. On the other hand, transient tissue hypoxia, consistent with I-R injury in vivo, enhances phosphorylated eNOS production while diminishing VEGF and iNOS. These data suggest that eNOS or iNOS derived from EPCs may play a critical role in recovery from different types of tissue ischemia.
eNOS or iNOS Derived From EPCs Plays a Differential Role in the Effect of IP
These findings suggested that the regulation of different NOS isoforms was different during the time course of hypoxic stress. Thus, to clarify the importance of eNOS or iNOS derived from EPCs after IP, we compared myocardial infarction size with or without IP followed by I-R or chronic ischemic injury among wild-type mice that were recipients of BMT from wild-type, eNOS–/–, or iNOS–/– mice, which resulted in chimeric mice in which only bone marrow–derived cells were deficient in expression of 1 of the NOS isoforms, whereas the myocardium retained normal NOS expression. In the case of the I-R injury model, the lack of iNOS in recruited EPCs significantly reduced the beneficial effect of IP (% reduction of infarct size –35.9±3.4% in wild-type BMT versus –22.5±3.3% in iNOS BMT, P<0.05), and IP actually aggravated ischemic injury in eNOS–/– BMT mice (% reduction of infarct size –35.9±3.4% in wild-type BMT versus 19.8±6.3% in eNOS BMT, P<0.0001; Figure 5a). These findings imply that eNOS, delivered from bone marrow–derived cells, plays a critical role in myocardial protection in the setting of I-R injury. On the other hand, in the case of the chronic ischemic injury model, with permanent coronary artery occlusion, the lack of eNOS in recruited EPCs significantly reduced the benefit of IP (% reduction of infarct size –29.4±3.2% in wild-type BMT versus –16.0±3.8% in eNOS BMT, P<0.05), and IP strikingly aggravated ischemic injury in iNOS–/– BMT mice (% reduction of infarct size –29.4±3.2% in wild-type BMT versus 62.2±6.8% in iNOS BMT, P<0.0001; Figure 5b). These findings imply that iNOS activity, recruited rapidly to the myocardium via bone marrow–derived EPCs, plays a significant role in myocardial protection in the setting of permanent coronary ischemia.
Discussion
These data suggest a novel mechanism for the protective effect of IP: the rapid deployment of reservoirs of NOS delivered to the site of injury via EPCs. IP alone is shown to rapidly modulate circulating EPC kinetics in the peripheral blood in association with the accumulation of EPCs in jeopardized myocardium. The EPCs then play 2 major roles in minimizing myocardial injury after prolonged, severe ischemia. EPCs directly preserve the microcirculation in ischemic myocardium via incorporation into vascular structures, consistent with prior data indicating this role for EPCs in ischemic tissue protection and recovery.35 The unexpected and perhaps more significant finding of these studies, however, is that EPCs recruited to the threatened myocardium play a unique role as repositories of NOS activities ie, eNOS and iNOS, as well as VEGF. Most striking, however, is the fact that the different NOS isoforms have different, perhaps contrasting effects depending on the type of ischemic insult. These activities of EPCs combine to help preserve the preexisting microcirculation indirectly and perhaps to thereby protect cardiomyocytes from ischemia-induced apoptosis and death.
The incorporation of EPCs in ischemic tissue has been shown to contribute to the recovery of ischemia through participation in neovascularization, both in animal studies and in promising early clinical trials.23,36 However, the other roles of EPCs in ischemic myocardium are not well understood, and controversy remains.29 In the present study, we demonstrate another role of EPCs as a favorable donor of NOS activity after IP. The mechanisms of the extremely rapid recruitment of EPCs and the strikingly different role for isoforms of NOS in different types of myocardial ischemia remain unsolved puzzles; however, the role of EPC-delivered NOS in myocardial protection is clear.
Recently, many investigators have documented the role of eNOS10,37 or iNOS18,38,39 in the cardioprotective effect of IP. Our data suggest that in the case of I-R injury after IP, activated eNOS produced from recruited EPCs plays an essential role in cardioprotection. A previous study reported that eNOS is not essential for the effect of IP37; however, this discrepancy can be explained by the use of a different experimental model, specifically an ex vivo isolated heart in a Langendorff perfusion system, thereby obviating the possibility of observing the in vivo phenomena induced by EPCs. Moreover, to the extent that we examined the expression of eNOS in the myocardium immunohistochemically, there was little activity noted in capillaries except for the endothelium in epicardial coronary arteries (data not shown). The major source of eNOS appeared to be bone marrow–derived EPCs. On the other hand, iNOS produced from recruited EPCs appears to play a crucial role in cardioprotection compared with eNOS in the setting of chronic ischemic injury after IP. Previous studies have documented a favorable role of iNOS in IP.18,38,39 In particular, Wang et al40 reported that IP upregulated iNOS in cardiomyocytes and played a protective role against myocardial damage. These authors showed iNOS mRNA expression in ischemic myocardium followed IP, and diffuse iNOS signals were detected with in situ hybridization and immunohistochemistry in the cytoplasmic space of cardiomyocytes. These reports and recent reviews of NOS signaling in IP19,41 are consistent with the present data; however, previous investigators did not consider the association between eNOS or iNOS and IP from the perspective of EPC contribution. These data are also consistent, from a more general perspective, with the concept put forth by Vasa et al42 that EPC phenotype is a clinically relevant factor in determining cardiovascular outcomes.
The present study demonstrates the first evidence that EPCs can contribute to a favorable effect of IP, acting as eNOS, iNOS, and VEGF donors in ischemic myocardium. Furthermore, we also show the differential roles of eNOS and iNOS derived from recruited EPCs in I-R or chronic ischemic injury after IP. These data provide novel insights in this field in terms of understanding the mechanisms of IP and provide clues to possible therapeutic approaches to enhance myocardial protection in the setting of ischemic injury.
Acknowledgments
This study was supported in part by NIH grants (HL-53354, HL-60911, HL-63414, HL-63695, and HL-66957). We thank Mickey Neely and Deirdre Costello for secretarial assistance, Hong Ma and Marianne Kearney for expert histological assistance, and Hiromi Nishimura for helpful discussions.
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Regenerative Medicine (H.N., T.A.), Institute of Biomedical Research and Innovation, Kobe, Japan.
Abstract
Background— The function of bone marrow–derived endothelial progenitor cells (EPCs) in repair of ischemic tissue has been the subject of intense scrutiny, and the capacity of these cells to contribute significantly to new blood vessels remains controversial. The possibility that EPCs could act as reservoirs of cytokines has been implied by several observations; however, a specific role for cytokine delivery has not been identified.
Methods and Results— We performed a series of experiments that revealed the rapid recruitment of EPCs to the myocardium by very short periods of ischemia, so-called ischemic preconditioning. The recruited EPCs express an array of potentially cardioprotective cytokines including nitric oxide synthase isoforms. Bone marrow transplantation studies, using donor marrow null for nitric oxide synthase isoforms, revealed that both endothelial and inducible nitric oxide synthase derived from bone marrow cells play essential roles in the cardioprotective effect that normally occurs after ischemic preconditioning.
Conclusions— These findings provide novel insights about the role of bone marrow–derived cells in ischemic preconditioning and also reveal that distinct mechanisms regulate recovery after ischemia-reperfusion and chronic ischemic injury.
Key Words: myocardial infarction ; ischemia ; nitric oxide synthase ; blood cells
Introduction
The notion that repetitive transient sublethal interruption of the blood supply to the myocardium, resulting in ischemia, followed by restoration of the blood supply, or reperfusion (ischemia-reperfusion [I-R]), can have a protective effect against subsequent prolonged ischemia has been established by clinical observation and experimental studies over the past 2 decades.1–4 The myocardial protection that results from ischemic preconditioning (IP) is characterized by reduced infarct size, decreased incidence of fatal arrhythmias, and reduced postischemic contractile cardiac dysfunction.4–6 Multiple mechanisms have been suggested to play a role in IP,7–10 including vascular endothelial growth factor (VEGF)–mediated neovascularization during myocardial ischemia11–13 protein kinase C activation,2,4,13–16 and nitric oxide synthase (NOS) activity,17–19 among others.9,20
Angiogenesis has also been shown to play a central role in the recovery of the myocardium after ischemia and infarction.12,13 We and others have identified endothelial progenitor cells (EPCs) in adult human peripheral blood21,22 and have shown that EPCs accumulate in active angiogenic foci and participate in neovascularization after ischemic insults, a concept consistent with postnatal vasculogenesis.23–25 Moreover, studies have shown that augmentation of the supply of bone marrow–derived progenitor cells improves outcome after ischemic injury.26–28 Nevertheless, the actual contribution of EPC-derived vessel formation to improved outcome has been controversial,29 and there has been a suggestion that the extent of physiological benefit that results from augmentation of the EPC supply in sites of ischemia has been greater than the apparent anatomic contribution of these cells in the newly formed vasculature.
Accordingly, we performed a series of investigations to test the hypothesis that EPCs could exert a protective effect in the setting of myocardial ischemia via the rapid delivery of cytokines. We used the model of IP because this simple maneuver has been shown to have dramatic protective effects on myocardial preservation and has been associated with the expression of a number of potentially protective cytokines. Initial pilot studies revealed a strikingly rapid recruitment of EPCs after transient IP and relatively minor EPC-mediated neovascularization compared with the degree of cardioprotection.
Methods
Mice and Bone Marrow Transplantation Model
Male FVB/NJ or C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me) aged 4 to 6 weeks were used as recipients for bone marrow transplantation (BMT). Transgenic mice of FVB/N-TgN (Tie-2-LacZ) 182Sato, eNOS–/– mice (B6.129P2-Nos3tmlUnc), or iNOS–/– mice (B6.129P2-Nos2tmlLau; Jackson Laboratories) were used as donors for the BMT. All subsequent procedures were performed on BMT mice. St. Elizabeth’s Institutional Animal Care and Use Committee approved this research protocol, including all procedures and animal care.
The procedure of BMT was performed as described previously.23,24 Briefly, the background/recipient mice were lethally irradiated for bone marrow ablation with 9.0 Gy for FVB/NJ mice and 12.0 Gy for C57BL/6J mice; each received 1 half million donor bone marrow mononuclear cells. At 6 to 8 weeks after BMT, by which time the bone marrow of the recipient mice was reconstituted, all the procedures described below were performed. Hearts of BMT mice were harvested at the indicated time points after surgery for histology.
Surgical Procedure
The mouse model of myocardial ischemia was based on that described previously.5,7 Briefly, after induction of anesthesia and mechanical ventilation, a left thoracotomy was performed in the fourth intercostal space, followed by pericardiectomy. An 8-0 monofilament nylon suture was passed under the left anterior descending coronary artery (LAD) just proximal to the first diagonal branch. A 5-mm section of polyethylene suture was placed on top of the LAD to secure the ligation without damaging the artery. Both ends of the suture were passed through a segment of flared PE10 tubing to form a snare. IP was induced by pulling the snare and clamping tube with 4 cycles of 4 minutes of LAD occlusion and 4 minutes of reperfusion. I-R injury was induced with 45 minutes of LAD occlusion, and chronic ischemic injury was induced with permanent LAD ligation. Control mice underwent thoracotomy and pericardiectomy followed by the passing of a suture under the LAD without interruption of blood flow for 30 minutes as a sham operation for comparison with IP.
Immunohistochemistry
The hearts of BMT mice were harvested at predetermined times after surgery and prepared for frozen tissue sectioning. Double-fluorescent immunohistochemistry was performed with an antibody against ;-galactosidase (;-gal) and endothelial NOS (eNOS), inducible NOS (iNOS), or VEGF. Nonspecific protein binding was blocked with 10% normal horse serum. Sections were incubated with rabbit polyclonal anti-;-gal antibody (1:250, Cortex) at 4°C overnight, followed by Cy3-goat anti-rabbit IgG (1:500, Jackson ImmunoResearch) as a secondary antibody for 30 minutes. Goat polyclonal anti-eNOS, -iNOS and -VEGF antibodies (1:250, Santa Cruz) and Cy2-donky anti-goat IgG (1:500, Jackson ImmunoResearch) were used as a secondary primary and its secondary antibody, respectively. Normal rabbit or goat IgG served as negative controls. The endothelial cell–specific marker FITC-isolectin-B4 (1:100; Vector Laboratories) was used for capillary staining. Nuclei were counterstained with DAPI (1:5000, Sigma), and sections were mounted in aqueous mounting medium. Images were examined with a fluorescent microscope (Nikon ECLIPSE TE200).
EPC Culture Assay and Bone Marrow–Derived EPC Culture
EPC culture assay and bone marrow–derived EPC culture was performed as described previously.24,30 Briefly, mononuclear cells isolated from 500 μL of peripheral blood were cultured in 5% FBS/EBM-2 (Clonetics) medium with supplements (SingleQuot Kit; Clonetics) on rat vitronectin (Sigma) with 0.1% gelatin-coated 4-well glass chamber slides. After 4 days in culture, cells were coincubated with DiI-acLDL (Biomedical Technologies) for 1 hour, followed by FITC-BS-1 lectin staining. The dual-stained cells, considered EPCs, were counted in 10 randomly selected high-power fields under a fluorescent microscope. Bone marrow–derived mononuclear cells of isolated tibia and femur were plated on cell culture dishes coated with rat vitronectin at a density of 5x105/cm2 and cultured in 5% FBS/EBM-2 medium. After 4 days in culture, nonadherent cells were removed, and adherent cells were reseeded at a density of 5x104/cm2. After 3 days in further culture, the cells were used as EPC-rich cell population for Western blot analysis.
Western Blot Analysis
Western blot analysis was performed as described previously.31 Briefly, cells cultured in EBM-2 medium with supplements under certain culture conditions were lysed in sample buffer in a 35-mm culture dish. Protein extracts were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane (BioRad). The membranes were blocked with 10% nonfat dry milk, then immunoblotted overnight at 4°C with rabbit polyclonal antibodies against mouse eNOS (1:1000, BD Pharmingen), phospho-eNOS (1:1000, Cell Signaling), iNOS (1:1000, BD Pharmingen), and VEGF (1:500, Santa Cruz) and a goat polyclonal antibody against -actin (1:1000, Santa Cruz). After 45 minutes in incubation with horseradish peroxidase–goat anti-rabbit IgG (1: 5000, Santa Cruz), immunoreactive bands were visualized with ECL reagent (Amersham). Densitometric analyses for the blots were performed with NIH Image software.
Statistical Analysis
Data are presented as mean±SEM. Significance (P<0.05) was determined by ANOVA followed by post hoc analysis with the Fisher procedure.
Results
EPCs Are Recruited to the Myocardium Immediately After IP Alone
To investigate EPC kinetics in peripheral blood and the myocardium after IP alone, we performed EPC culture assays and histological evaluations in Tie-2/LacZ BMT mice, which receive bone marrow from transgenic mice that constitutively express ;-gal regulated by the promoter of the endothelial gene, Tie-2.32,33 As shown in Figure 1a, the number of circulating EPCs decreased immediately after IP to a significantly greater extent than in a sham control group (n=6 at the indicated time points, respectively; 33.2±3.3 EPCs per high-power field [HPF] in cell culture assay at baseline, 21.5±1.9 EPCs/HPF in sham versus 11.7±1.3 EPCs/HPF in IP at 1 hour, P<0.01). Circulating EPC counts were then similar for the next few hours (sham=8.7±1.4 EPCs/HPF versus IP=8.3±0.8 EPCs/HPF at 3 hours, P=NS; sham=16.0±1.5 EPCs/HPF versus IP=19.8±2.7 EPCs/HPF at 6 hours, P=NS) and then increased significantly in the IP group, peaking at 3 days (21.2±1.7 versus 33.7±3.5 EPCs/HPF at 18 hours, 32.0±3.3 versus 42.7±4.7 EPCs/HPF at 1 day, and 41.5±1.4 versus 59.1±1.6 EPCs/HPF at 3 days; P<0.01). By day 7, the levels in both groups had returned toward baseline levels, although they remained higher in the IP group (29.7±4.2 versus 37.8±2.4 EPCs/HPF, P<0.01). To assess the microvascular architecture, in situ fluorescent staining with the endothelial cell–specific marker FITC-conjugated BS1-lectin (Vector Laboratories) was performed with minor modification as described previously.24 Briefly, 10 minutes after systemic injection of BS1-lectin (0.1 mg per mouse), hearts were fixed with 2% paraformaldehyde for 2 hours and prepared for frozen cross-sectioning. Immunofluorescent staining revealed that the early, precipitous decrease in circulating EPCs in the IP group coincided with rapid EPC recruitment to the jeopardized zone of the myocardium. As shown in Figure 1b, EPCs, identified by red fluorescence resulting from ;-gal immunoreactivity, appeared in the myocardium immediately, 1 and 3 hours after IP, and gradually diminished thereafter.
These studies revealed that repetitive transient LAD occlusion and reperfusion induced dramatic recruitment of EPCs into the ischemic zone of the myocardium immediately after IP alone. This was accompanied by a biphasic modulation of EPC kinetics in the peripheral circulation, with a rapid decrease in circulating EPCs that coincided with the appearance of EPCs in the myocardium. Thus, EPC kinetics in peripheral blood and recruitment of EPCs in the myocardium suggested a dynamic equilibrium. Subsequently, the increased number of EPCs in the late phases after IP may have resulted from EPC mobilization by VEGF (or other cytokines) released by the ischemic myocardium (data not shown). Although a sham operation, including thoracotomy and pericardiectomy, can also affect EPC kinetics, this more modest response is thought to be due to the wound-healing process.23 The implication is that recruitment of bone marrow–derived cells to sites of injury, previously referred to under the rubric of "inflammation," may comprise a variety of responses, with some targeted directly to the mechanisms of tissue repair or vascular preservation. Indeed, most of the recruited cells in the myocardium immediately after IP were not inflammatory cells expressing markers of CD3 and CD13 (data not shown) but were Tie-2–expressing cells, consistent with EPCs.
IP Reduces Infarct Size and Preserves Capillary Density After I-R Injury
Next, we sought to document the extent of myocardial protection by IP, and its relationship to the recruitment of EPCs, by inducing myocardial infarction by prolonged ischemia followed by reperfusion (I-R) with or without preceding IP. Consistent with the pilot studies of IP alone, the number of ;-gal–expressing cells (indicating bone marrow origin and Tie-2 expression) in the peri-infarct area was significantly higher in the IP group than in the sham group (10.6±2.4 versus 43.0±4.9/mm2, P<0.01; Figure 2a). Also as expected, the extent of myocardial damage induced by I-R injury was decreased in the IP group (n=5) compared with the sham group (n=5) at 7 days. IP significantly reduced infarct size (2.4±0.2 mm2 in the sham group versus 1.0±0.2 mm2 in the IP group; 58% reduction, P<0.01; Figure 2b). Representative photographs of immunofluorescent staining with in situ perfusion of BS1-lectin (green) to identify capillaries in the ischemic area are shown in Figure 3a. Capillary density (in 3 random HPFs) in the ischemic area 3 days after I-R injury was significantly greater in the IP group than in the sham group, which suggests that IP had preserved the microvasculature (101.5±16.8/HPF versus 239.0±7.8/HPF, P<0.01; Figure 3b). The participation of EPCs, indicated by double staining of red (;-gal, ie, bone marrow–derived Tie-2–expressing cells) and green (endothelium of any origin), also was observed in capillaries of the myocardium (Figure 3a). The extent of incorporated EPCs in the microvasculature of the ischemic area was also significantly higher in the IP group than in the sham group (25.5±2.1/HPF versus 58.3±5.2/HPF, P<0.01; Figure 3c). This indicates that bone marrow–derived cells had incorporated into the myocardial microvasculature in greater numbers when ischemia-induced injury was preceded by IP.
Incorporated EPCs Produce eNOS, iNOS, and VEGF in Ischemic Myocardium
Double fluorescent immunostaining in ischemic myocardium 3 days after surgery in the IP group revealed that the incorporated ;-gal–expressing EPCs (red) and some of the cardiomyocytes in ischemic myocardium expressed eNOS, iNOS, and VEGF (green; Figure 4a). Merged images reveal large numbers of double-positive cells expressing ;-gal (Tie-2–expressing cells of bone marrow origin) and eNOS, iNOS, and VEGF, respectively, which reveals that the EPCs were significant sources of cardioprotective factors.
To clarify the regulation of EPC-produced cytokines in ischemic myocardium, we next performed an in vitro study using cultured EPCs under hypoxic conditions in an attempt to mimic myocardial ischemia. EPCs isolated from bone marrow were cultured under normoxic (95% air and 5% CO2) or hypoxic (<2% oxygen, 95% N2 and 5% CO2)34 conditions in a standard incubator at 37°C. Cells were then further exposed to either hypoxia or normoxia 24 hours after hypoxia for the indicated time. In Western blot analyses, EPC phospho-eNOS abundance was gradually and slightly reduced by persistent hypoxia (arbitrary units, 1.0 in normoxia versus 0.6±0.10 after 24 hours of hypoxia, P<0.05; Figure 4b), whereas expression of iNOS and, to a lesser extent, VEGF was significantly induced (iNOS, 0 in normoxia versus 0.2±0.02 at 8 hours and 1.3±0.15 at 24 hours in hypoxia, P<0.0001; VEGF, 1.0 in normoxia versus 1.4±0.12 at 8 hours and 1.4±0.17 at 24 hours in hypoxia, P<0.01; Figures 4c and 4d). In contrast, hypoxia-induced repression of eNOS expression was gradually reversed in normoxia (arbitrary units, 1.0 in 24 hours of hypoxia versus 1.5±0.09 after 8 hours and 1.8±0.06 after 24 hours in normoxia, P<0.001; Figure 4b). Similarly, the upregulation of iNOS and VEGF expression induced by hypoxia was significantly reversed by normoxia (iNOS, 1.0 in 24 hours of hypoxia versus 0.6±0.09, 0.3±0.06, and 0 after 4, 8, and 24 hours in normoxia, respectively, P<0.0001; VEGF, 1.0 in 24 hours of hypoxia versus 0.9±0.04 and 0.5±0.04 after 8 and 24 hours in normoxia, P<0.05 and <0.0001, respectively; Figures 4c and 4d). Although these data may not necessarily reflect changes in gene expression by EPCs in the myocardium, they do provide a starting point for our investigation of the potential mechanisms involved.
These findings show that EPC expression of the active form of eNOS, phospho-eNOS, appears to be diminished by hypoxia. Conversely, iNOS in EPCs is induced by hypoxia, as is endogenous VEGF.34 The upregulation of iNOS and VEGF is also reversed by normoxia. Hypoxia promotes EPC migration but inhibits EPC mitogenic activity in response to VEGF (data not shown).34
To integrate these results, EPCs recruited to the ischemic myocardium are stimulated by the local tissue environment and upregulate expression of NOS and VEGF. Sustained tissue hypoxia, which reflects chronic ischemic injury in vivo, not only promotes migration activity of EPCs but also further enhances EPC VEGF production and iNOS expression. On the other hand, transient tissue hypoxia, consistent with I-R injury in vivo, enhances phosphorylated eNOS production while diminishing VEGF and iNOS. These data suggest that eNOS or iNOS derived from EPCs may play a critical role in recovery from different types of tissue ischemia.
eNOS or iNOS Derived From EPCs Plays a Differential Role in the Effect of IP
These findings suggested that the regulation of different NOS isoforms was different during the time course of hypoxic stress. Thus, to clarify the importance of eNOS or iNOS derived from EPCs after IP, we compared myocardial infarction size with or without IP followed by I-R or chronic ischemic injury among wild-type mice that were recipients of BMT from wild-type, eNOS–/–, or iNOS–/– mice, which resulted in chimeric mice in which only bone marrow–derived cells were deficient in expression of 1 of the NOS isoforms, whereas the myocardium retained normal NOS expression. In the case of the I-R injury model, the lack of iNOS in recruited EPCs significantly reduced the beneficial effect of IP (% reduction of infarct size –35.9±3.4% in wild-type BMT versus –22.5±3.3% in iNOS BMT, P<0.05), and IP actually aggravated ischemic injury in eNOS–/– BMT mice (% reduction of infarct size –35.9±3.4% in wild-type BMT versus 19.8±6.3% in eNOS BMT, P<0.0001; Figure 5a). These findings imply that eNOS, delivered from bone marrow–derived cells, plays a critical role in myocardial protection in the setting of I-R injury. On the other hand, in the case of the chronic ischemic injury model, with permanent coronary artery occlusion, the lack of eNOS in recruited EPCs significantly reduced the benefit of IP (% reduction of infarct size –29.4±3.2% in wild-type BMT versus –16.0±3.8% in eNOS BMT, P<0.05), and IP strikingly aggravated ischemic injury in iNOS–/– BMT mice (% reduction of infarct size –29.4±3.2% in wild-type BMT versus 62.2±6.8% in iNOS BMT, P<0.0001; Figure 5b). These findings imply that iNOS activity, recruited rapidly to the myocardium via bone marrow–derived EPCs, plays a significant role in myocardial protection in the setting of permanent coronary ischemia.
Discussion
These data suggest a novel mechanism for the protective effect of IP: the rapid deployment of reservoirs of NOS delivered to the site of injury via EPCs. IP alone is shown to rapidly modulate circulating EPC kinetics in the peripheral blood in association with the accumulation of EPCs in jeopardized myocardium. The EPCs then play 2 major roles in minimizing myocardial injury after prolonged, severe ischemia. EPCs directly preserve the microcirculation in ischemic myocardium via incorporation into vascular structures, consistent with prior data indicating this role for EPCs in ischemic tissue protection and recovery.35 The unexpected and perhaps more significant finding of these studies, however, is that EPCs recruited to the threatened myocardium play a unique role as repositories of NOS activities ie, eNOS and iNOS, as well as VEGF. Most striking, however, is the fact that the different NOS isoforms have different, perhaps contrasting effects depending on the type of ischemic insult. These activities of EPCs combine to help preserve the preexisting microcirculation indirectly and perhaps to thereby protect cardiomyocytes from ischemia-induced apoptosis and death.
The incorporation of EPCs in ischemic tissue has been shown to contribute to the recovery of ischemia through participation in neovascularization, both in animal studies and in promising early clinical trials.23,36 However, the other roles of EPCs in ischemic myocardium are not well understood, and controversy remains.29 In the present study, we demonstrate another role of EPCs as a favorable donor of NOS activity after IP. The mechanisms of the extremely rapid recruitment of EPCs and the strikingly different role for isoforms of NOS in different types of myocardial ischemia remain unsolved puzzles; however, the role of EPC-delivered NOS in myocardial protection is clear.
Recently, many investigators have documented the role of eNOS10,37 or iNOS18,38,39 in the cardioprotective effect of IP. Our data suggest that in the case of I-R injury after IP, activated eNOS produced from recruited EPCs plays an essential role in cardioprotection. A previous study reported that eNOS is not essential for the effect of IP37; however, this discrepancy can be explained by the use of a different experimental model, specifically an ex vivo isolated heart in a Langendorff perfusion system, thereby obviating the possibility of observing the in vivo phenomena induced by EPCs. Moreover, to the extent that we examined the expression of eNOS in the myocardium immunohistochemically, there was little activity noted in capillaries except for the endothelium in epicardial coronary arteries (data not shown). The major source of eNOS appeared to be bone marrow–derived EPCs. On the other hand, iNOS produced from recruited EPCs appears to play a crucial role in cardioprotection compared with eNOS in the setting of chronic ischemic injury after IP. Previous studies have documented a favorable role of iNOS in IP.18,38,39 In particular, Wang et al40 reported that IP upregulated iNOS in cardiomyocytes and played a protective role against myocardial damage. These authors showed iNOS mRNA expression in ischemic myocardium followed IP, and diffuse iNOS signals were detected with in situ hybridization and immunohistochemistry in the cytoplasmic space of cardiomyocytes. These reports and recent reviews of NOS signaling in IP19,41 are consistent with the present data; however, previous investigators did not consider the association between eNOS or iNOS and IP from the perspective of EPC contribution. These data are also consistent, from a more general perspective, with the concept put forth by Vasa et al42 that EPC phenotype is a clinically relevant factor in determining cardiovascular outcomes.
The present study demonstrates the first evidence that EPCs can contribute to a favorable effect of IP, acting as eNOS, iNOS, and VEGF donors in ischemic myocardium. Furthermore, we also show the differential roles of eNOS and iNOS derived from recruited EPCs in I-R or chronic ischemic injury after IP. These data provide novel insights in this field in terms of understanding the mechanisms of IP and provide clues to possible therapeutic approaches to enhance myocardial protection in the setting of ischemic injury.
Acknowledgments
This study was supported in part by NIH grants (HL-53354, HL-60911, HL-63414, HL-63695, and HL-66957). We thank Mickey Neely and Deirdre Costello for secretarial assistance, Hong Ma and Marianne Kearney for expert histological assistance, and Hiromi Nishimura for helpful discussions.
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