BM Suppression Induces MMP-9 Expression in BM Cells
While BM suppression with 5-FU results in apoptosis of actively cycling HSCs and progenitor cells, it does not affect HSCs in G0 of cell cycle. This model is ideal for studying factors that promote recruitment of HSCs during hematopoietic reconstitution. We found an increase in pro-MMP-9 () and active MMP-9 (), but not tissue inhibitor of metalloproteinases (TIMP-1) in supernatants of BM cells of MMP-9 wild-type animals three days after 5-FU treatment. In BM cells of untreated animals, there was a small amount of MMP-9 () derived from resident neutrophils (Murphy et al., 1982
). In wild-type mice, BM hematopoietic and stromal cells expressed MMP-9 three days after treatment with 5-FU (). These data suggest that both pro-MMP-9 and MMP-9 are upregulated in BM cells after myelosuppression.
MMP-9 Is Induced in BM Cells after BM Ablation
MMP-9−/− Mice Show Delayed Hematopoietic Recovery Following 5-FU Treatment
To understand the role of MMP-9 in promoting HSC recruitment and hematopoietic recovery after BM suppression, we used MMP-9-deficientmice. Under steady-state conditions, adult MMP-9−/− and MMP-9+/+ mice have similar peripheral white blood cell (WBC) counts and both became leukopenic within six days after 5-FU treatment (). However, in 5-FU-treated MMP-9−/− mice, the recovery of WBC () and platelets (data not shown) was delayed by an additional 8 days. This prolonged delay in hematopoietic recovery resulted in the death of 72% (n = 16, p < 0.001) of 5-FU-treated MMP-9−/− mice, while all of MMP-9+/+ mice survived (). These data suggest that MMP-9 plays a critical role in accelerating hematopoietic reconstitution.
Delayed Hematopoietic Recovery and Increased Mortality in MMP-9−/− Mice after Myelosuppression
Cell cycling, motility, and differentiation of HSCs are accelerated during hematopoietic recovery after BM ablation. We next determined whether the impaired hematopoietic recovery of HSCs in MMP-9−/−
mice was due to an alteration in the stem/progenitor cell cycling status. Hematopoietic cells phenotypically marked as Lin−
comprise a large percentage of long-term repopulating HSCs (Cheshier et al., 1999
). Under steady-state conditions, the percentage and the total number of Sca-1+
() and Lin−
cells () in S phase of cell cycle were not significantly different in MMP-9+/+
mice. However, following 5-FU myeloablation, the number of these cells entering S phase of cell cycle was impaired in MMP-9−/−
mice. These data suggest that although during steady-state hematopoiesis there is no difference in the number of cycling repopulating cells, in the recovery phase after myelosuppression, lack of MMP-9 results in a diminished recruitment of cycling cells leading to profound BM hypocellularity.
Restoration of Myeloid and Megakaryocytic Lineages Is Impaired in MMP-9−/− Mice
On day 6 after 5-FU treatment, in MMP-9+/+ mice, BM cellularity increased and hematopoietic cell clusters were seen in close contact to the bone “osteoblastic zone (O),” followed by a shift of cells moving toward the blood vessel-enriched “vascular zone (V)” on day 10 (). However, in the BM of MMP-9−/− animals, there was a paucity of hematopoietic cell clusters in both the osteoblastic and vascular zones, even on day 10 after 5-FU treatment. By day 10, the proportion of differentiated CD11b+Gr-1+ myeloid () and vWF+ megakaryocytic precursor cells () in the BM of 5-FU-treated MMP-9−/− mice was dramatically reduced. The impaired recruitment of repopulating stem and progenitor cells into the vascular zone and the lack of differentiation into myeloid and megakaryocytic lineages in MMP-9−/− mice led us to hypothesize that MMP-9 may exert its effects through the release of a stem cell-active cytokine.
Recruitment and Differentiation of Hematopoietic Cells Are Impaired in MMP-9−/− Mice
Release of Soluble KitL (sKitL) Is Impaired in MMP-9−/− Mice after BM Suppression
Among the known stem cell-active chemokines and cytokines, KitL conveys signals that modulate survival, adhesion, and motility of c-Kit+
HSC and endothelial cells. sKitL is expressed as a membrane (mKitL) form and can be cleaved to a soluble form (sKitL) (Huang et al., 1992
). Indeed, plasma levels of sKitL increased 3-fold during the hematopoietic regeneration phase in MMP-9+/+
mice, peaking at 6 days when there was active proliferation in the BM. In contrast, baseline sKitL plasma levels were very low in MMP-9−/−
mice and did not increase following BM ablation (). These data suggest that MMP-9 promotes rapid release of sKitL facilitating BM recovery.
MMP-9 Mediated Release of sKitL Enhances Hematopoietic Reconstitution
Unstimulated BM stromal cells express both mKitL and MMP-9 (Heinrich et al., 1993
; Marquez-Curtis et al., 2001
). Although MMP-9 was present at low levels in the supernatants of the unstimulated murine BM stromal cell line MS-5, which expresses mKitL, addition of recombinant MMP-9 rapidly promoted the release of sKitL. This shedding was blocked by a synthetic metalloproteinase inhibitor (MPI; ). These data suggest that MMP-9 can effectively promote the release of sKitL from stromal cells.
However, MMP-9 expression is upregulated in both hematopoietic and stromal compartments of the BM after myeloablation (). To identify which cellular compartment is more critical in providing bioactive MMP-9 following myelosuppression, we developed chimeric mice by transplanting BM cells from MMP-9−/−
mice into lethally irradiated MMP-9+/+
recipients resulting in chimeric mice in which hematopoietic cells are MMP-9 deficient. Transplantation and engraftment of BM from MMP-9+/+
mice resulted in mice with MMP-9-deficient stroma (Supplemental Figure S4a
, available at http://www.cell.com/cgi/content/full/109/5/625/DC1
). As controls, MMP-9+/+
BM cells were transplanted into lethally irradiated MMP-9+/+
mice and MMP-9−/−
BM cells into lethally irradiated MMP-9−/−
mice. All mice showed engraftment after 16 days as determined by WBC counts. The majority of MMP-9+/+
recipients transplanted with either MMP-9−/−
BM or MMP-9−/−
recipients transplanted with MMP-9+/+
BM survived 5-FU myelosuppression. As expected, high mortality was observed in the MMP-9−/−
group transplanted with MMP-9−/−
BM cells. These data indicate that MMP-9 expressed either by stromal or hematopoietic cells is sufficient to support hematopoiesis after myelosuppression.
Exogenous sKitL Restores Hematopoietic Recovery and Mobilization in MMP-9−/− Mice
Under steady-state conditions, elevated sKitL levels delivered by adenoviral vector expressing sKitL (AdsKitL) increased WBC in both MMP-9−/−
mice (). Progenitor mobilization determined by either the frequency of mobilized Sca-1+
cells () or colony-forming unit cells (CFU-Cs) in the peripheral blood (Supplemental Figure S4b
at above URL) was enhanced in the MMP-9−/−
mice after AdsKitL introduction.
Since MMP-9 augments the release of sKitL, we next asked whether the delayed hematopoietic recovery seen in MMP-9−/− mice could be restored directly by exogenous recombinant sKitL. Treatment of MMP-9−/− mice with recombinant sKitL following 5-FU treatment resulted in rapid recovery of WBC (). In addition, sKitL treatment following 5-FU BM suppression resulted in a rapid increase in BM cellularity and hematopoietic reconstitution in MMP-9−/− mice () and restored survival of MMP-9−/− mice (decreasing mortality to 17% compared to 72% observed in the PBS group, n = 10 in each group). These data suggest that MMP-9 mediated release of sKitL is essential for promoting stem cell differentiation, accelerating hematopoietic reconstitution following BM ablation.
Chemokines Released upon BM Suppression Promote MMP-9 Expression
But what regulates MMP-9? One clue came from the observation that the chemokine stromal cell-derived factor-1 (SDF-1) increased after myelosuppression (Ponomaryov et al., 2000
). Plasma levels of SDF-1 increased following 5-FU treatment, peaking on day 8 (). These data suggest that rapid elevation of chemo/cytokine levels after BM ablation contributes to MMP-9 upregulation, setting the stage for HSC recruitment.
Chemo/Cytokines Induce MMP-9 Expression in BM Hematopoietic Cells
To determine whether chemo/cytokines induce MMP-9 expression in vivo, we introduced SDF-1, VEGF, and G-CSF into wild-type mice. BM of the mice treated with SDF-1, VEGF, or G-CSF showed increased immunoreactive MMP-9 on stromal and hematopoietic cells (). These studies raised the possibility that chemo/cytokine-induced MMP-9 activation mediates mobilization of hematopoietic cells.
We found that SDF-1 and VEGF stimulate the release of pro-MMP-9 and induce migration of human CD34+ progenitor and stem cells (5 week cobblestone forming cells [CAFC] and long-term culture initiating cells [LTC-IC]) in a transwell migration assay (). The migration of CD34+ cells was completely blocked by addition of MPIs (CGS 27023A and 5-phenyl-1,10-phenanthroline). Direct incubation of CD34+ cells with MPIs in suspension culture did not alter their proliferation in CAFC or LTC-IC assays (data not shown). These data suggest that chemokines induce the functional expression of MMP-9 on CD34+ cells and their migration.
Chemokine-Induced Mobilization of BM Repopulating Cells Is Impaired in MMP-9-Deficient Mice
Plasma elevation of SDF-1, VEGF, and G-CSF in MMP-9+/+ mice, but not in MMP-9−/− mice mobilized mature WBCs () and hematopoietic progenitors (CFU-Cs; ). The mobilization of hematopoietic cells into the circulation followed the kinetics of plasma elevation of these chemokines (). We next determined if MMP-9 plays a role in mobilization of cells with repopulating capacity by transplanting peripheral blood mononuclear cells (PBMCs) mobilized by G-CSF as the source of BM repopulating cells into lethally irradiated syngeneic mice. Mice transplanted with G-CSF-mobilized PBMCs harvested from MMP-9+/+ mice 5 days after chemokine treatment showed long-term donor cell engraftment and survival, whereas those transplanted with G-CSF-mobilized PBMCs from MMP-9−/− mice died during the 20 days allowed for engraftment (). These data show that MMP-9 plays a role in mobilization of BM repopulating cells.
Chemo/Cytokine-Induced HSC Mobilization Is Impaired in MMP-9−/− Mice
The necessity of chemokine-induced mobilization on MMP activity was confirmed by administrating a synthetic MPI in vivo, which also decreased WBC counts and blocked chemokine-induced mobilization in MMP-9-competent mice (data not shown). Importantly, mobilization of hematopoietic cells with short-term repopulating potential (CFU-S) was completely blocked in MPI-treated mice (Supplemental Figure S6a
available at http://www.cell.com/cgi/content/full/109/5/625/DC1
). Lethally irradiated syngeneic mice transplanted with SDF-1-, VEGF-, and G-CSF-mobilized PBMCs showed long-term engraftment in 55%, 40%, and 80% of these mice, respectively (Supplemental Figures S6b, c, and d
at above URL). Similar to MMP-9−/−
mice, transplantation of PBMCs from MPI-treated mice failed to reconstitute hematopoiesis in lethally irradiated recipients and all recipient mice died within 19 days.
SDF-1 and VEGF Increase sKitL Plasma Levels
If a significant role of MMP-9 activation in the postmyelosuppression process is the release of sKitL, then impaired hematopoietic cell mobilization following SDF-1 and VEGF treatment of MMP-9−/− mice could be due to their inability to produce sKitL. Indeed, we observed increased plasma sKitL levels in MMP-9+/+ mice after treatment with SDF-1, VEGF, and G-CSF as compared to untreated animals (). This chemo/cytokine-induced upregulation of sKitL was impaired in MMP-9−/− mice. These results highlight the importance of MMP-9-induced sKitL release for hematopoietic recovery.
Mobilization of BM-Derived Endothelial Progenitors Is MMP-9 Dependent
c-Kit is expressed on BM-derived progenitors that can give rise to cardiac muscle, skeletal muscle, and endothelial cells. Therefore, we asked if MMP-9 plays a more general role in regulating other tissue-specific stem cells. Elevation of plasma levels of SDF-1 and VEGF in wild-type mice increased the mobilization of circulating endothelial progenitors (CEPs) as assessed by the generation of late outgrowth endothelial cell colony forming units (CFU-EC) and the expression of VEGF-receptor-2 (VEGFR2; ). In contrast, VEGF did not mobilize CFU-EC or VEGFR2+ cells into the circulation of wild-type mice treated with MPI (). Similarly, VEGF increased the number of CFU-EC in the PB () and VEGFR2+ cells in the BM of MMP-9+/+, but not in MMP-9−/− mice (). Collectively, these results suggest that MMP-9 activation is required for the recruitment and mobilization of BM-derived CEPs.
Endothelial Cell Progenitor Mobilization Is Blocked in MMP-9−/− Mice