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Published as doi: 10.1096/fj.07-8636com.
 (The FASEB Journal. 2008;22:930-940.)
© 2008 FASEB
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(The FASEB Journal. 2008;22:930-940.)
© 2008 FASEB

Activation of c-kit is necessary for mobilization of reparative bone marrow progenitor cells in response to cardiac injury

Shafie S. Fazel*, Liwen Chen*, Denis Angoulvant*, Shu-Hong Li*, Richard D. Weisel*, Armand Keating{dagger} and Ren-Ke Li*,1

* Division of Cardiac Surgery, Department of Surgery, Toronto General Hospital Research Institute, and

{dagger} Department of Medical Oncology and Hematology, Princess Margaret Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada

1Correspondence: Toronto General Hospital, MaRS Centre, Toronto Medical Discovery Tower, Rm. 3–702, 101 College St., Toronto, ON, Canada, M5G 1L7, E-mail: renkeli{at}uhnres.utoronto.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiovascular disease is the number-one cause of mortality in the developed world. The aim of this study is to define the mechanisms by which bone marrow progenitor cells are mobilized in response to cardiac ischemic injury. We used a closed-chest model of murine cardiac infarction/reperfusion, which segregated the surgical thoracotomy from the induction of cardiac infarction, so that we could study isolated fluctuations in cytokines without the confounding impact of surgery. We show here that bone marrow activation of the c-kit tyrosine kinase receptor in response to released soluble KitL is necessary for bone marrow progenitor cell mobilization after ischemic cardiac injury. We also show that release of KitL and c-kit activation require the activity of matrix metalloproteinase-9 within the bone marrow compartment. Finally, we demonstrate that mice with c-kit dysfunction develop cardiac failure after myocardial infarction and that bone marrow transplantation rescues the failing cardiac phenotype. In light of the ongoing trials of progenitor cell therapy for heart disease, our study outlines the endogenous repair mechanisms that are invoked after cardiac injury. Amplification of this pathway may aid in restoration of cardiac function after myocardial infarction.—Fazel, S. S., Chen, L., Angoulvant, D., Li, S. H., Weisel, R. D., Keating, A., Li, R. K. Activation of c-kit is necessary for mobilization of reparative bone marrow progenitor cells in response to cardiac injury.


Key Words: myocardial infarction • heart failure • stem cells • cell signaling


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PRESENT THERAPY OF MYOCARDIAL infarction (MI) is directed toward early reperfusion by reopening the acutely occluded coronary artery. No established therapy exists to enhance cardiac repair after myocardial infarction/reperfusion (MI/R), and frequently the damage to the myocardium is irreversible. Direct myocardial cellular therapy and/or bone marrow mobilizing cytokine therapy are undergoing investigation for the treatment of MI/R (1) . Evidence suggests that bone marrow or circulating progenitor cell therapies after MI/R improve cardiac function (2 3 4 5) , regardless of whether transdifferentiation of the cells to cardiomyocytes occurs (6 7 8) or does not (9 10 11) . Cytokine mobilization of the appropriate cells from the bone marrow may be a more effective method of increasing progenitor cells within the coronary circulation. However, safety concerns were raised when global bone marrow mobilization with granulocyte colony-stimulating factor led to a 70% risk of adverse vascular remodeling (12) . Understanding the molecular mechanisms that induce progenitor cell mobilization in response to cardiac ischemia may provide the necessary road map to develop therapeutic targets for specific progenitor cell mobilization without induction of a general proinflammatory state.

A robust cardiac repair process, which is largely responsible for mechanically stabilizing the infarcted area, is activated after MI/R. The principal cells involved in this process are the recruited inflammatory cells and the cardiac endothelial and fibroblast populations (13) . We recently described the novel and critical role of the bone marrow c-kit-expressing cells in promoting cardiac repair (14) . In that study, we documented that, after MI, bone marrow c-kit+ VEGF-R2+ Sca-1lo CD45dull cells were mobilized, and that in the absence of their mobilization two important components of cardiac repair were severely attenuated: 1) an angiostatic ratio of angiopoietin-1 to angiopioetin-2 and diminished peri-infarct vascular endothelial cell growth factor expression prevented rapid endothelial proliferation, and 2) the myofibroblast response to MI was markedly diminished. In combination, the absence of vessel-rich repair tissue caused a precipitous decline in cardiac function. These results, which were recapitulated when a pharmacologic inhibitor of c-kit function, imatinib mesylate, was used, have been confirmed by others (15) . The data taken together suggest that the c-kit+ cells are responsible for transitioning the myocardium from inflammation to the repair phase. We postulated that the beneficial impact of cellular-based therapeutics is to facilitate this transition by increasing the number of such cells within the myocardium.

Recent human data confirm that MI/R induces the mobilization of bone marrow progenitor cells (16) . These cells express the tyrosine kinase receptor, c-kit, which has been directly (6 , 7 , 17 18 19) or indirectly (9 , 20 , 21) implicated in cardiac repair and/or regeneration. After binding its ligand KitL, the c-kit receptor is autophosphorylated (22 , 23) . Activation of the c-kit receptor is necessary for the mobilization of the c-kit+ hematopoietic stem cells (24 25 26) . It has been speculated that activation of the c-kit receptor is also necessary for the mobilization of c-kit+ endothelial progenitor cells (EPCs) (27 , 28) . KitL is present in two isoforms, the membrane-bound isoform (mKitL) and a soluble isoform (sKitL). Post-translational cleavage of mKitL at the cell surface can release sKitL. Both mKitL and sKitL may bind c-kit; however, the signaling mechanism differs. For instance, the Steel-Dickie mouse, which is deficient in mKitL, has a defect in the hematopoietic supportive microenvironment within the bone marrow despite physiological levels of sKitL and is unable to accept bone marrow transplantation (29) . Cleavage of mKitL to sKitL may occur by proteolytic enzymes (30) . A class of proteolytic enzymes capable of cleaving mKitL is the matrix metalloproteinases (MMP), whose activity is kept in check by the tissue inhibitors of matrix metalloproteinases (TIMP) (31) . Indeed, activation of the c-kit+ hematopoietic stem cells in response to myeloablative therapy induced by 5-fluorouracil requires the activity of MMP-9 (26) . The impact of MI/R on the molecular mechanism of EPC mobilization and the related kinetics are not known.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and materials
The 8- to 10-wk-old female C57Bl6 mice were purchased from Charles River Laboratories (Senneville, QC, Canada). The 8- to 10-wk-old FVB.Cg-Mmp9tm1Tvu/J MMP-9-deficient mice, WBB6F1-KitW/KitW-v c-kit mutant, and Kit+/+ congenic controls were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The W mutation is the result of a 78-amino acid deletion that includes the transmembrane domain of the c-kit protein, and the W-v mutation is a missense mutation in the kinase domain of the c-kit coding sequence (32) . The mmp-9 null mice were made by replacing exon 2 and all of intron 2 with a PGK-driven promoter (33) .

Animal procedures
The Animal Care Committee of the University Health Network approved all experimental procedures according to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86 -23, revised 1996).

Animals were sedated, intubated, ventilated with a closed-chest myocardial ischemia-reperfusion model (Harvard Apparatus, Holliston, MA, USA), and maintained with 2% isoflurane. Through a thoracotomy, the heart was visualized. A 7–0 prolene suture was passed through the chest wall, through a 2 mm segment of 25-gauge angiocatheter, and looped around the left coronary artery and back through the angiocatheter and the chest wall. The chest wall then was closed, suture ends were tucked under the skin flaps, and the skin was re-approximated. Seven days later, animals were sedated and maintained using a nose cone. A 5 mm segment of the previous skin incision was opened and the suture ends were located. Under electrocardiographic monitoring, tension was placed on the suture ends to produce ST-segment elevation. After 60 min, the suture was cut, and ST-segment normalization was documented in every instance. A similar technique has been described previously (34) . Permanent coronary ligation was performed as described (14) .

To determine the area of necrotic myocardium, mice were sacrificed 24 h after injury, and their hearts were transversely cut into 1 mm thick sections. The myocardial rings were then incubated in 1% triphenyltetrazolium chloride (TTC) at 37°C for 30 min.

Bone marrow reconstitution: female KitW/KitW-v mice were irradiated (950 rads). Then, 2.5 x 107 fresh bone marrow cells from male Kit+/+ donors were injected into the tail vein (Kit+/+->KitW/KitW-v bone marrow chimeric mice). After 6 wk, reconstitution was assessed by PCR for the SRY Y-chromosome gene using bone marrow genomic DNA preparations.

Cardiac function
For echocardiography, two-dimensional images were obtained using a 15 MHz linear array probe and a Sequoia echocardiography system (Siemens AG, Munich, Germany) as described (35) .

EPC assay
The technique of EPC quantification from mice has been described previously (36) . The total number of EPCs was counted in 5 random fields at x400 in a blinded fashion by 2 investigators.

In vitro human umbilical vein endothelial cell (HUVEC) assay
HUVECs were cultured in EBM. After trypsinization, the cells were plated at a density of 1 x 105 cells/ml in 6-well plates. Human recombinant TNF-{alpha} was added at concentrations of 2, 20, and 200 ng/ml in triplicates. After incubation for 1, 3, or 9 h, RNA was isolated using the Trizol reagent. Reverse transcriptase-polymerase chain reaction (RT-PCR) was then performed.

Colony-forming unit (CFU) assay
On days 0, 1, 3, and 7, the harvested hearts were divided into the injured (area subtended by the ligated coronary artery) and noninjured regions. Minced tissue was then digested in 0.2% collagenase and spun through Iscove’s modified Dulbecco medium containing 10% FBS. Then, 3 x 106 cells were plated in duplicates in Methocult medium. After 7–10 days, the number of colonies with >50 cells per colony was counted in a blinded fashion by 2 investigators using an Olympus phase-contrast microscope (Olympus America, Melville, NY, USA).

Gelatin zymography
The bone marrow supernatant was incubated with gelatin-agarose beads overnight. After elution with sodium dodecyl sulfate, the samples were analyzed using PAGE containing 0.1 mg/ml gelatin. The gels then were incubated 15 min at room temperature in 2.5% Triton X-100 and then at 37°C overnight in 20 mM NaCl, 5mM CaCl2, 0.02% Brij-35, and 50 mM Tris-HCl buffer (pH 7.6), stained with 0.1% Coomassie Brilliant Blue R-250, and destained in 10% acetic acid/30% methanol in H2O.

RT-PCR and immunoblotting
Total RNA was extracted from bone marrow cells by TriZol reagent as per manufacturer’s instructions. RNA quantity and quality were determined by spectrophotometry at A260 and A280. RT-PCR and immunoblotting techniques were performed as described previously (14) . Primer sequences are available on request.

Microscopy
Immunohistochemistry and confocal microscopy techniques have been described previously (14) . Slides were examined using a LSM510 META (Carl Zeiss, New York, NY, USA) confocal microscope. Negative controls where the primary antibodies were omitted were used to set the laser power and detector gains prior to scanning.

Statistics
Data are presented as mean ± SE. Statistical analysis between the two groups was carried out using a two-tailed t test with Welch’s correction for nonequal variance. Time course analyses were carried out using two-way ANOVA, unless otherwise indicated. If the overall difference was significant, posthoc multiple comparisons with Bonferroni’s correction were performed. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Closed chest MI/R model
One hour of coronary ligation caused extensive necrosis of the subtended myocardium (Fig. 1 A), and the injured segment underwent fibrosis (Fig. 1C ). Over 6 wk, MI/R caused progressive ventricular dilation (Fig. 1B ) and dysfunction (Fig. 1D ). The differential impact of MI/R vs. MI alone is compared in Fig. 1 , panels B and D. To characterize the impact of thoracotomy alone on inflammatory cytokine expression and EPC mobilization, animals were studied over 7 days. Thoracotomy alone caused a rapid increase in serum IL-1β and IL-6 but not TNF-{alpha}. In the bone marrow, thoracotomy alone caused an increase in MMP-2 and MMP-9 activity but did not result in the release of EPCs in the peripheral circulation. All of the above-noted alterations returned to baseline by day 7 after thoracotomy. Therefore, we thereafter studied only animals that were subjected to MI/R 7 days after thoracotomy to appreciate the specific response to cardiac ischemia without the confounding impact of surgery on the systemic cytokine milieu.


Figure 1
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  		Figure 1. Closed chest MI/R model. A) One hour of coronary ligation results in significant myocardial necrosis, as demonstrated by triphenyltetrazolium chloride staining of 1 mm heart sections from base to apex (right to left). B) Permanent coronary ligation (MI) causes more ventricular dilation than myocardial infarction/reperfusion (MI/R) after 6 wk. C) Top panel: masson trichrome staining showing myocardium distal to the coronary artery ligature. Macroscopic evidence of myocardial injury decreases in size over the ensuing 7 days (D7) as the injured region begins to thin. Bottom panel: hematoxylin and eosin staining of the injured region demonstrates significant myocardial cell loss as seen by day 3 (D3). By day 7 (D7), there is apposition of the injured region borders with significant fibrosis. D) Echocardiography data showing worse ventricular dilation and function in MI vs. MI/R group at 6 wk. LVEDA = left ventricular end diastolic area; LVESA = left ventricular end systolic area; AFC = area fractional contraction (n=5 in each group). *P < 0.05 vs. Sham; **P < 0.05 vs. MI/R. E) Serum inflammatory cytokines, bone marrow MMP-2 and -9, and peripheral circulation EPC quantification after thoracotomy alone were studied over a time course showing that significant fluctuations occur with surgery alone (n=3–5 in each group per time point). *P < 0.05 vs. D0.

MI/R causes rapid soluble KitL up-regulation in the bone marrow
Both SDF-1{alpha} (37 , 38) and KitL (26 , 28) have been shown to be involved in bone marrow progenitor cell mobilization. We evaluated the impact of isolated cardiac ischemic injury on the systemic and bone marrow concentrations of KitL and SDF-1{alpha}. We observed that KitL increased 1.3-fold (P=0.06) (Fig. 2 A) and SDF1-{alpha} increased 1.5-fold (P=0.02) (Fig. 2B ) in the plasma. In the bone marrow, MI/R caused a 2.2-fold increase (P=0.04) in KitL level by day 1 (Fig. 2C ) whereas SDF-1{alpha} levels rose gradually to day 7 (Fig. 2D ). Thus, early after MI/R, bone marrow KitL and not SDF-1{alpha} levels spike.


Figure 2
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  		Figure 2. Soluble cytokine levels after MI/R in plasma and bone marrow aspirates. Results from 3–4 animals per group per time point are demonstrated.*P < 0.05.

MI/R decreases membrane KitL but increases soluble KitL, resulting in c-kit phosphorylation
We next examined the cause of the increase in soluble KitL at the mRNA and protein level in the bone marrow. We found that MI/R did not significantly affect transcription of the KitL gene up to 7 days after injury (Fig. 3 A). However, MI/R caused an early significant decrease in membrane isoform of KitL by day 1 that returned to baseline by day 7 (D0, 100±0%; D1, 64±5.5%; D7, 111.5±15.9%; ANOVA P=0.03) (Fig. 3B ). Concomitantly, a marked increase in soluble isoform of KitL was detected by immunoblotting (P=0.01) (Fig. 3B ), suggesting conversion of one isoform to another. Within 1 day of injury, the c-kit protein was phosphorylated, returning to baseline status by day 7 (Fig. 3C ), suggesting that the oscillations in KitL isoforms had functional consequences.


Figure 3
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  		Figure 3. KitL oscillations in response to MI/R. A) MI/R did not result in changes in KitL gene transcription assayed by RT-PCR. M = markers; H = water control; SP = KitL plasmid +ve control. B) One day (D1) after MI/R, membrane KitL decreased. Concomitantly, soluble KitL (lower molecular weight band) increased within the bone marrow. These oscillations returned to baseline by day 3 (representative blot from 5 independent experiments). C) Immunoblotting for phosphorylated c-kit (Phos. c-kit) and c-kit, demonstrating activation of the c-kit receptor on bone marrow cells on D1 after MI/R (representative blot from 3–5 independent experiments).

MI/R causes bone marrow MMP-9 up-regulation
To understand the underlying fluctuations in MMPs that may be responsible for conversion of membrane to soluble KitL, we evaluated bone marrow MMP-2, MMP-9, TIMP-1, and TIMP-3 at the transcriptional, translational, and protein activity levels. Cardiac injury resulted in rapid up-regulation of MMP-9 but not MMP-2, TIMP-1, or TIMP-3 mRNA (Fig. 4 A). Accordingly, MMP-9 protein level increased in both the cellular and noncellular fractions of the bone marrow (Fig. 4B ) within 24 h of MI/R (protein levels: D0, 100±0%; D1, 241.3±19.6%; D7, 135.3±38.1%; ANOVA P=0.01). Protein levels of MMP-2, TIMP-1, and TIMP-3 did not change (Fig. 4B ). Using gelatinolysis as a measure of MMP-9 bioactivity, we confirmed that bone marrow extracellular tissue also had higher MMP-9 activity after cardiac injury (Fig. 4C ). MMP-2 activity remained constant and well below the magnitude of MMP-9 activity (Fig. 4C ).


Figure 4
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  		Figure 4. Up-regulation of MMP-9 after MI/R. A) RT-PCR demonstrated increased MMP-9 mRNA levels but no change in MMP-2, TIMP-1, and TIMP-3 mRNA levels (representative of 3 independent experiments). M = markers; H = water control. B) By immunoblotting, we observed significant up-regulation of MMP-9 (representative of 5 independent experiments) but not MMP-2, without changes in TIMP-1 and TIMP-3 in bone marrow cells (representative of 3 independent experiments). C) Gelatin zymography of bone marrow supernatant, showing an increase in MMP-9 gelatinolytic activity but not in MMP-2 activity levels. The two right lanes represent MMP-2 and MMP-9 positive controls, respectively (representative zymogram of 5 independent experiments). +2 = MMP-2 positive control; +9 = MMP-9 positive control.

Oscillations in bone marrow KitL are MMP-9-dependent
To establish a causal relationship between MMP-9 and c-kit activity, we investigated the response to MI/R in MMP-9–/– mice compared to C57Bl/6 wild-type mice. We confirmed again that in response to MI/R in C57Bl/6 mice, MMP-9 activity was upregulated (Fig. 5 A, left two lanes), membrane KitL was decreased, and c-kit was phosphorylated (Fig. 5B , left two lanes). In contrast, in MMP-9–/– animals, no MMP-9 activity was detectable and there was no compensatory increase in MMP-2 activity (Fig. 5A , right six lanes). Moreover, membrane KitL levels did not decrease in response to MI/R (Fig. 5B , right six lanes). Soluble KitL levels did not change either in the bone marrow or in the plasma of MMP-9–/– mice (Fig. 5C ). As a result, the c-kit protein remained in an unphosphorylated state (Fig. 5B , right six lanes). Thus, after cardiac injury, MMP-9 activity is necessary for c-kit activation in the bone marrow.


Figure 5
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  		Figure 5. Activation of c-kit is dependent on MMP-9 activity. A) Gelatin zymogram demonstrating MMP-9 activity before (D0) and 1 day (D1) after MI/R. MMP-9 activity is increased after MI/R in C57Bl/6 (left two lanes) but not in mmp-9–/– (right six lanes) mice, which completely lack MMP-9. There is no compensatory increase in MMP-2 level or activity. +2 = MMP-2 positive control; +9 = MMP-9 positive control. B) Immunoblotting for KitL, Phos. c-kit, c-kit, and actin. Unlike in C57Bl/6 mice (left two lanes), MI/R in mmp-9–/– mice (right six lanes) fails to decrease membrane KitL and fails to lead to c-kit phosphorylation. C) Soluble KitL levels quantified by enzyme-linked immunosorbent assay, showing that MI/R fails to increase soluble KitL levels both in the bone marrow and in the plasma in mmp-9–/– animals.

MMP-9 and c-kit activity are required for EPC mobilization
We next examined EPC mobilization in response to MI/R. In C57Bl/6 mice, the number of detectable peripheral EPCs increased to maximal levels by day 1 and returned to baseline by day 7 after MI/R (Fig. 6 A, B), mirroring the bone marrow soluble KitL levels.


Figure 6
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  		Figure 6. EPC mobilization in response to MI/R. A) Number of splenic mononuclear cells that are able to attach to basement membrane protein (fibronectin)-coated plates over 48 h and go on to exhibit phenotypic characteristics of mature endothelial cells (uptake DiI-labeled [red] acetylated low-density lipoprotein and bind fluoroisothiocyanate-conjugated [green] lectin) is increased after MI/R. These cells are thought to be representative of the endothelial progenitor cell population (36) . B) Quantified data on the number of endothelial progenitor cells obtained from counting yellow cells in above assay in five x400 blind fields. After MI/R, rapid increase in the number of such cells is observed (representative of 3 independent experiments). C) Genomic DNA PCR for the sex-determining region Y chromosome (sry) gene to determine the degree of chimerism in female irradiated KitW/KitW-v mice that had received male bone marrow cells from Kit+/+ mice via the tail vein. In lanes 2–6, increasing proportions of male-into-female cell population "standard curve" were used to gauge degree of chimerism. Lanes 7–11 are bands obtained from 5 randomly chosen animals 6 wk after irradiation and bone marrow cell transplantation. Degree of chimerism was greater than 70%. M = markers. D) Number of EPCs before and 24 h after MI/R in the experimental group of mice (3–6 per group). *P < 0.05.

To investigate the role of the c-kit activation in EPC mobilization, we used the c-kit mutant KitW/KitW-v mouse. Bone marrow cells of this mouse expressed the c-kit receptor, but the mean fluorescence was 0.2 log units lower than their congenic Kit+/+ littermates. Whereas the C57Bl6 and Kit+/+ bone marrow cells phorphorylated c-kit when cultured in the presence of 50 ng/ml recombinant KitL, the KitW/KitW–v bone marrow cells did not. To examine whether this phenotype could be rescued with prior transplantation of Kit+/+ bone marrow cells into KitW/KitW-v mice, we lethally irradiated KitW/KitW-v mice. Injection of Kit+/+ bone marrow cells into conditioned hosts resulted in >70% bone marrow reconstitution in the KitW/KitW-v bone marrow 6 wk later (Fig. 6C ). The c-kit phosphorylation in response to KitL was restored in the bone marrow cells of the chimeric mouse.

In response to cardiac ischemia, the number of peripheral EPCs increased 3.4-fold (P<0.001) in the Kit+/+ mouse (Fig. 6D ). Consistent with the alteration in the c-kit pathway documented above, the increase in EPCs was abrogated in MMP-9–/– animals (Fig. 6D ). Likewise, the KitW/KitW-v mouse did not mobilize EPCs. Baseline peripheral EPCs were increased in the Kit+/+ -> KitW/KitW-v bone marrow chimeric group. Cardiac MI/R caused a significant further increase in peripheral EPCs by 1.6-fold at 24 h, proving that c-kit function within the bone marrow compartment is necessary for EPC mobilization (Fig. 6D ) into the peripheral circulation.

MMP-9 and c-kit activity are required for progenitor cell trafficking to the heart
Next, we evaluated the impact of MI/R on progenitor cell trafficking to the heart using a functional hematopoietic colony formation assay (Fig. 7 A). The number of progenitor cells that could be detected in the heart increased within the injured region but not the noninjured region of the heart in the C57Bl/6 mice (Fig. 7B ). No significant infiltration of hematopoietic progenitor cells was seen in the MMP-9–/– or the KitW/KitW-v mice (Fig. 7C ). Progenitor cell trafficking was restored in the Kit+/+ -> KitW/KitW-v bone marrow chimeric group (Fig. 7C ) with a 4.2-fold rise 24 h after MI/R (P=0.04).


Figure 7
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  		Figure 7. Hematopoietic progenitor cell recruitment to the heart. A) After collagenase digestion of the infarcted and noninfarcted regions of the heart, the isolated cells were plated on hematopoietic progenitor cell-supporting semisolid medium, and hematopoietic colonies were observed 7–10 days after plating. Representative phase-contrast images at low magnification of colonies are shown. BFU-E = blast-forming unit-erythroblast; CFU-preB = colony forming unit lymphoid pre B; CFU-GM = colony forming unit granulocyte macrophage; CFU-GEMM = colony forming unit granulocyte erythrocyte megakaryocyte macrophage. B) Number of colonies were quantified in both the infarcted and the noninfarcted segment of C57Bl/6 hearts over a time course (n=3 per time point). Hematopoietic progenitor cell colonies were most abundant on day 3 after MI/R and were present only in the infarcted region. C) Number of hematopoietic progenitor colonies before and 24 h after MI/R in the injured region of the heart in the various experimental groups (n=3–6 per group). *P < 0.05.

The injured segment of myocardium attracts and supports c-kit-expressing progenitor cells
We next examined the response of the injured myocardium to ischemic injury. We found that KitL mRNA was upregulated within the injured segment. This rise in KitL mRNA was significantly different from the unaffected KitL mRNA quantity in the noninjured segment (Fig. 8 A). Changes in mRNA levels also reflected changes at the protein level detected by immunoblotting. Specifically, within the injured region of the heart, the KitL protein level increased to a peak on day 3 after MI. Again, the KitL protein level in the noninjured segment was unaffected by the ischemic injury. Then we sought to determine the source of myocardial KitL. Immunohistochemistry showed that venular endothelial cells within the injured region had marked expression of KitL as compared to venules in the noninjured region (Fig. 8B ). The up-regulation of KitL in the myocardium was coincident with infiltration of c-kit+ cells into the peri-infarct zone as demonstrated by 3-color confocal microscopy (Fig. 8C ) and flow cytometry (Fig. 8D ). Consistent with the heightened KitL levels within the peri-infarct area, the majority of the c-kit+ cells that were detected were noted to have phosphorylation of the c-kit receptor (Fig. 8C ), strongly suggesting that the KitL levels were sufficient to cause c-kit signaling. We have shown previously that the c-kit+ cells detected in infarcted myocardium are from the bone marrow (14) .


Figure 8
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  		Figure 8. Cardiac ischemic injury leads to increased expression of SCF in the injured segment. A) Hearts were harvested at various time points after MI/R and divided into the injured and nonjured segments. These segments then were used for RT-PCR (top 2 rows) or for immunoblotting (bottom 2 rows). Results from 3 independent experiments are quantified in the right panel. Both SCF mRNA and protein levels were markedly increased within the injured segment of the heart. B) Immunohistochemistry of the injured and noninjured segment of the heart showed that SCF is expressed principally by the endothelium of large venule-like structures. C) Triple-color confocal microscopy (blue=nuclei; green=actin; red=c-kit or phosphorylated [P] c-kit) demonstrated infiltration of cells expressing c-kit and containing P c-kit molecule after MI/R into the peri-infarct border zone. D) Flow cytometry of single-cell suspension from the infarcted region showed infiltration of c-kit-expressing cells that peaked 3 days after injury (2.4±0.6%) (n=3 per time point). E) Incubation of HUVECs with increasing concentrations of TNF-{alpha} resulted in the up-regulation of KitL gene expression, as measured by RT-PCR in the linear range (representative of 3 independent experiments).

Considering that KitL up-regulation was not apparent immediately after ischemic injury, we postulated that KitL up-regulation was not an ischemia-specific response but rather caused by the inflammatory reaction within the damaged myocardium. We, therefore, tested whether TNF-{alpha}, a potent inflammatory cytokine highly expressed by infarcted myocardium, would cause KitL up-regulation in HUVECs. HUVECs were cultured in varying concentrations of recombinant TNF-{alpha}. TNF-{alpha} reproducibly caused a significant up-regulation of KitL transcript by 9 h of exposure (Fig. 8E ).

Abnormal function of bone marrow c-kit receptor results in rapid cardiac failure
We have described previously (14) the precipitous cardiac failure in the KitW/KitW-v compared to Kit+/+ mice after MI and the rescue of the phenotype by transplantation of bone marrow cells from the Kit+/+ into the KitW/KitW-v (Table 1 ). These results confirm the functional importance of c-kit function in the mobilization of progenitor cells to the heart after ischemic injury.


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  		Table 1. The importance of c-kit function on bone marrow cells in cardiac repair after ischemic injury, as measured by transthoracic echocardiography


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previously we have shown that the KitW/KitW-v mice progress rapidly to dilated cardiomyopathy after coronary ligation when compared to their Kit+/+ congenic wild-type littermates (14) . In this article we show that c-kit activation by an MMP-9-dependant pathway is necessary for the mobilization of bone marrow progenitor cells to the heart. We observed that MMP-9 activity is responsible for the cleavage of membrane KitL into soluble KitL, which is then available to bind its receptor, c-kit. The resulting phosphorylation of the c-kit receptor corresponds to the mobilization of peripheral EPCs. In both MMP-9–/– (no cleavage of membrane KitL to soluble KitL) and KitW/KitW-v animals, these changes in response to cardiac injury were not observed, and trafficking of progenitor cells to the heart was markedly diminished. The phenotype in KitW/KitW-v mice was rescued by prior bone marrow transplantation from Kit+/+ mice.

We used a model of closed-chest ischemic myocardial injury to segregate surgical trauma from the isolated effects of myocardial ischemia on systemic cytokines. Our preliminary studies, as supported by the work of others, showed that significant cytokine fluctuations occur with surgical trauma alone. Thus, models in which cytokines and cell trafficking patterns are observed require that the effect of surgery alone is controlled. Biochemical evidence of significant myocardial necrosis was present in all cases in which ST-segment elevation by electrocardiographic monitoring during the procedure was observed, as supported by the investigations of others (34 , 39) .

A notable aspect of the present report is that we document the direct crosstalk between the damaged heart and the bone marrow progenitors. Although mobilization of mature bone marrow cells to the injured heart is a well-described phenomenon (40) , the mature bone marrow cells, with the exception of mast cells, do not express c-kit. Thus, the observed oscillations in KitL level that resulted in the phosphorylation of c-kit represent direct crosstalk aimed at the progenitor population within the bone marrow and/or the mast cells (41) .

One such population that has been implicated in myocardial regenerative efforts is the EPC, which also expresses the c-kit receptor (42) . EPCs are thought to actively partake in angiogenesis by both direct incorporation into newly forming vessels (27 , 36) and by elaborating angiogenic cytokines (43 44 45) in both physiological and pathological scenarios (46) . Absence of EPC mobilization leads to decreased infarct angiogenesis, poor ventricular function, and inefficient infarct repair, as we observed previously.

The requirement for MMP-9 in progenitor cell mobilization is also supported by previous work that demonstrated MMP-9 to be necessary for the mobilization of c-kit+ hematopoietic stem cells in response to myeloablative therapy (26) . We speculate that MMP-9, by cleaving membrane KitL to soluble KitL, renders KitL available to c-kit binding on EPCs, as suggested also by recent studies involving hemangiocytes (47) . Presumably, in the unstimulated bone marrow, the KitL is spatially separated from its receptor. Likewise, although KitL cleavage and c-kit phosphorylation were an acute event, the change in absolute magnitude of total MMP-9 activity was relatively small. Also, surgery alone also caused a spike in MMP-9 without increasing the number of peripheral EPCs. We hypothesize that MMP-9, in the absence of ischemic injury, is sequestered from cells that express membrane KitL. Both direct bone marrow injury (26) and, in our case, distant ischemic myocardial injury, led to increased MMP-9 mediated conversion of membrane KitL to soluble KitL. The mechanism of MMP-9 up-regulation in the correct spatiotemporal sequence after ischemic injury is unknown.

The impact of progenitor cell mobilization on cardiac function after MI could not be evaluated in the MMP-9–/– model. MMP-9, independent of its effects in the bone marrow, is critical for post-MI cardiac remodeling through its effects on extracellular matrix degradation and angiogenesis (48 49 50) . Various bone marrow-derived cells are known to express MMP-9, such as neutrophils, monocyte/macrophages, and mast cells (33 , 51) . The upstream signal that causes increased bone marrow MMP-9 activity may range from cytokine bioactivation to neuroendocrine-mediated activation (52 , 53) . The short time interval between injury and full mobilization is much more rapid than previously reported and is likely to represent the physiological nature of the injury: cardiac ischemia vs. 5-fluorouracil administration (26) or low-dose total-body irradiation (52) .

Administration of KitL along with G-CSF improves cardiac function by enhancing endogenous cardiac repair that may have become inefficient in old age (54 , 55) . Expression of KitL has been described previously in a canine but not a rodent model of cardiac ischemia (41 , 56) . Endothelial cells have been known to express KitL (57 , 58) , but its up-regulation in response to the inflammatory cytokine TNF-{alpha} has not been reported previously. Search of the region upstream of the KitL promoter by MatInspector software (www.genomatix.de) revealed 1 NF{kappa}B binding site on the human KitL gene and 2 NF{kappa}B binding sites on the murine KitL gene. TNF-{alpha} is expressed principally in the infarct and peri-infarct regions and is upregulated rapidly within 24 h after infarction. Its expression is maintained at least 35 days in the infarcted segment (59) . Coupling of KitL to TNF-{alpha} expression allows highest KitL expression in a spatiotemporal sequence, ideally suited to enhance cardiac repair.

In conclusion, marrow progenitor cell mobilization in response to cardiac ischemia requires MMP-9 and c-kit tyrosine kinase activity. To circumvent issues with adverse vascular remodeling, generalized proinflammatory milieu may be averted by direct activation of the c-kit+ marrow progenitor cells. Such a strategy may allow specific progenitor cell mobilization without the concomitant mobilization of mature bone marrow-derived inflammatory cells. However, the recent findings that aging adversely impacts the cardiac repair response that is normally accentuated by KitL raises major obstacles in both cytokine-based (55) and cellular-based (60) therapies for cardiovascular disease because the majority of patients with cardiovascular disease are advanced in age.


   ACKNOWLEDGMENTS
 
This work was supported by operating grants from the Canadian Institute of Health Research (CIHR; MOP14795) to R.K.L. and S.F., Heart and Stroke Foundation (HSF; T5287) to R.D.W. and S.F., and the Physician Services Incorporated Foundation (R04-23) to S.F. S.F. is a CIHR and a joint CIHR/HSF TACTICS Research Fellow and a Medical Scientist Training Fellow of the McLaughlin Centre for Molecular Medicine.

Received for publication April 5, 2007. Accepted for publication September 27, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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