Impaired BM recovery and progenitor mobilization after irradiation in MMP-9–deficient mice
CEPs contribute to vessel formation at the ischemic site (15
). We initially asked whether IR releases hematopoietic progenitors and/or CEP into circulation in an MMP-dependent manner. Both MMP-9–deficient and WT mice tolerated sublethal IR at doses of 1.0, 2.0, 6.5, and 7.5 Gy (unpublished data), and showed no difference in the survival of a lethal IR dose (9.5 Gy). BM hematopoietic recovery after a sublethal dose of 6.5 Gy, as determined by BM cellularity ( A), the hematopoietic colony-forming unit cells (CFU-Cs) ( B), CFU endothelial cell (CFU-EC) recovery ( C), and peripheral blood cell count recovery ( D) was delayed in MMP-9−/−
mice compared with controls. These data are in accordance with our previous study in which we showed that MMP-9–mediated KitL release is essential for BM cell recovery, because the delayed BM recovery of MMP-9−/−
mice after 5-fluorouracil could be restored by soluble KitL (14
Figure 1. Irradiation-induced BM regeneration and progenitor mobilization are MMP-9–dependent and coincide with an increase in angiogenic factors. MMP-9+/+ and MMP-9−/− mice were irradiated with 6.5 Gy. (A) BM cell number (n = 8/group), (more ...)
When WT mice were irradiated with a sublethal dose of 6.5 Gy as total body IR (TBI), MMP-9 expression in supernatants of BM cells increased starting from day 7 until day 21 after IR ( E), while TIMP-1 expression did not change (not depicted). Supernatants of BM cells of unirradiated animals had only small amounts of pro– and active forms of MMP-9. Plasma levels of pro–MMP-9 also increased after IR (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20050959/DC1
). Immunoreactive MMP-9 was low in unirradiated mice ( F a) but was highly expressed 6 and 12 d after TBI in close contact with osteoblasts ( F, c and d). By day 30 after IR, MMP-9 staining was found in the central areas of the BM ( F, d). By contrast, MMP-2 expression showed no major change as determined by zymography (Fig. S1 B).
What is the role of increased MMP-9 activity in the BM endothelium after IR? Under steady-state conditions, the number of capillaries, as a direct measure of BM endothelium, was not different between MMP-9+/+ and MMP-9−/− mice. BM vascular recovery following IR was impaired in irradiated MMP-9−/− mice, as estimated by determining the number of capillaries (Fig. S1, C and D). The VEGFR1+ (Fig. S1 E) and VEGFR2+ cell fractions (Fig. S1 F) were lower in MMP-9−/− mice, and their recovery was delayed.
IR stimulates hematopoietic and endothelial progenitor mobilization
We observed enhanced mobilization of CFU-Cs ( G) and CFU-ECs ( H) into circulation in irradiated WT, but not in MMP-9−/−
mice. Which factors govern progenitor mobilization? We recently reported that plasma levels of stromal cell–derived factor 1 (SDF-1 or CXCL12) and KitL are elevated following 5-fluorouracil treatment (stress-hematopoiesis) (4
). Here, we show that IR increases stem cell–active mobilization factors, including plasma KitL ( I), VEGF ( J), SDF-1 (Fig. S1 G), and PlGF (Fig. S1 H) in a partly MMP-9–dependent manner.
IR at high doses might have adverse effects. If IR could ultimately be used to improve ischemia-related diseases, low-dose IR could have efficacy in progenitor recovery and growth factor production. Hematopoietic recovery following low-dose IR was delayed in MMP-9−/−
as compared with MMP-9+/+
mice, as assayed by the number of BM cells per femur ( A). We observed that post-IR–induced leukopenia was attenuated after an IR dose of 2 Gy ( B) in MMP-9−/−
mice. Significantly, low-dose IR induced MMP-9 expression in BM cells ( C) and augmented plasma KitL ( D), PlGF (Fig. S2 A, available at http://www.jem.org/cgi/content/full/jem.20050959/DC1
), and VEGF levels ( E) in an MMP-9–dependent manner. Moreover, circulating CFU-C and CFU-EC release (Fig. S2 B and C, available at http://www.jem.org/cgi/content/full/jem.20050959/DC1
) was increased after low-dose IR in MMP-9+/+
but not MMP-9−/−
Figure 2. Low-dose irradiation and HL ischemia induce progenitor mobilization. (A–C) MMP-9+/+ and MMP-9−/− mice were irradiated with 2 Gy. (A) BM cell number per femur following low-dose IR and (B) white blood cell recovery (n = 8) was determined (more ...)
To test the potential therapeutic pro-angiogenic effect of low-dose IR and its regulation by MMP-9, we chose a mouse model of hind limb (HL) ischemia. Operated MMP-9+/+
mice receiving an IR dose of 2 Gy did not show pad necrosis (unpublished data), whereas unirradiated controls showed necrotic changes. In muscle tissue of MMP-9+/+
mice, larger vessels stained positive for MMP-9 ( F) after IR (2 Gy). Kidney, lung, and spleen tissue showed no difference in MMP-9 staining before or after IR (unpublished data). HL ischemia increased mobilization of CFU-Cs ( G) and CFU-ECs ( H). However, HL ischemia, in combination with IR, resulted in a profound increase in CFU-Cs (up to 30-fold compared with steady-state values) and CFU-ECs, which coincided with peaks in plasma levels of VEGF ( I), KitL (Fig. S2 D, available at http://www.jem.org/cgi/content/full/jem.20050959/DC1
), and PlGF (Fig. S2 E) in MMP-9+/+
, but not in MMP-9−/−
mice. Plasma VEGF, KitL, and PlGF levels rose further (two to threefold) after IR in these mice ( I; and Fig. S2, D and E). Soluble factors known to be involved in ischemic regeneration (16
)—such as monocyte chemotactic protein 1 (MCP-1; Fig. S2 F), transforming growth factor β (TGF-β; Fig. S2 G), and basic fibroblast growth factor (unpublished data)—showed slight increases following HL ischemia induction, but were identical in MMP-9+/+
mice. Importantly, IR did not further increase the plasma levels of these factors in either group of mice.
To understand which cell types increase after IR, we analyzed peripheral blood mononuclear cells (PBMCs) via FACS (Becton Dickinson) using mAbs against c-kit and VEGFR-2 after IR. No differences were found in the number of VEGFR−
cells following HL ischemia or after IR. HL ischemia mobilized VEGFR-2+
cells, most likely comprised of ECs (18
) in MMP-9+/+
mice, but to a lower extent in MMP-9−/−
mice ( J). IR further augmented the release of VEGFR-2+
cells into circulation in a MMP-9–dependent manner. Importantly, IR increased the VEGFR-2+
cell population in MMP-9+/+
but not MMP-9−/−
mice. Toluidine blue O+
cells were the major constituent of the VEGFR-2+
fraction ( J).
Low-dose IR stimulates blood vessel formation in ischemic limbs
Revascularization of the ischemic limb was macroscopically delayed in MMP-9−/− mice (unpublished data), with fewer von Willebrand factor (vWF)-positive capillaries, resulting in significantly more necrosis compared with MMP-9+/+ mice (). In addition, the number of vessels covered with smooth muscle cells (SMCs) was higher in HL ischemia–induced MMP-9+/+ mice compared with MMP-9−/− animals ( A).
Figure 3. Low-dose IR-mediated revascularization is delayed in MMP-9−/− mice. (A–E) HL ischemia was induced in MMP-9+/+ and MMP-9−/− mice. Mice then received a single low-dose TBI (2 Gy) or no IR. (A) Muscle sections from (more ...)
After IR, HL ischemia–induced MMP-9+/+ mice showed few signs of necrosis compared with unirradiated controls (). Muscle tissue from HL ischemia–induced MMP-9−/− mice showed vast areas of necrotic tissue. IR only slightly decreased the necrotic area in these animals. IR under ischemic conditions increased the number of capillaries in MMP-9+/+ mice but only slightly in MMP-9−/− mice ( C). Restoration of vascular function as measured by thermography () and laser Doppler () paralleled the vascular regeneration. Ten days after induction of HL ischemia, laser Doppler analysis revealed decreased blood flow in HL ischemia–induced MMP-9−/− compared with MMP-9+/+ mice ( E). IR of HL ischemia–induced MMP-9+/+ mice increased both mean temperature () and blood flow () compared with nonirradiated controls. In contrast, IR of HL ischemia–induced MMP-9−/− mice only slightly improved mean temperature () and blood flow (). Upon histological examination, necrotic area, density of vWF+ vessels, and perfusion of irradiated animals without induction of ischemia was not different in irradiated compared with unirradiated muscle tissue in MMP-9+/+ or MMP-9−/− mice (). These data suggest that low-dose IR alone does not cause visible changes in the muscle microenvironment, but applied under ischemic conditions stimulates angiogenesis.
IR increases the number of tissue-resident mast cells
The increase in Toluidine blue–positive mast cell–like cells in circulation following IR ( J) led us to hypothesize that IR improves mast cell migration into ischemic tissue. A higher number of mast cells was found in ischemic muscle tissue from MMP-9+/+ compared with MMP-9−/− mice ( A, a–d). Mast cells were identified as Toluidine blue– and (unpublished data) mast cell tryptase–positive cells. Using adjacent tissue sections, we detected VEGF mRNA in mast cells via in situ hybridization ( A, e–h), implying that these cells are the major source of VEGF in the tissues after ischemia and IR. Staining of adjacent sections with VEGFR-2 mAb revealed that both mast cells and ECs stained positive for VEGFR-2 ( A, i–l). IR further augmented the number of mast cells in the muscle tissue of MMP-9+/+ mice but not MMP-9−/− mice ( A, a–d, m).
Figure 4. MMP-9–dependent release of VEGF by mast cells and of KitL by stromal cells promotes mast cell migration, a necessary requirement for ischemic regeneration. (A, B) HL ischemia was induced in MMP-9+/+ and MMP-9−/− mice, and mice (more ...)
Because mice had been exposed to TBI, we determined the number of mast cells in other tissues. The number of Toluidine blue–positive mast cells was higher 14 d after low-dose IR in the skin ( B, a and b) of MMP-9+/+ mice, but not MMP-9−/− mice, compared with untreated controls. As a measure of mast cell activation, we determined histamine levels in irradiated and unirradiated WT mice. IR increased plasma histamine levels threefold as compared with unirradiated controls (Fig. S2 H).
Mast cell–mediated VEGF release after low-dose irradiation mediates angiogenesis and is impaired in MMP-9–deficient mice
KitL and VEGF promote mast cell migration (19
), and mast cells show baseline MMP-9 activity. We therefore examined whether the decreased number of mast cells in MMP-9−/−
mouse tissue was due to a migratory defect of mast cells toward KitL and/or VEGF. In a transwell migration assay, KitL and VEGF induced migration of MMP-9+/+
mast cells ( C) but not MMP-9−/−
mast cells. VEGF-mediated migration of MMP-9+/+
mast cells was partially blocked by neutralizing antibodies against VEGFR-1 or VEGFR-2 but was completely blocked by addition of an MMP inhibitor (MPI CGS 27023A).
PT18 cells (mast cell line) and MMP-9+/+ mast cells showed baseline VEGF secretion ( D), which was significantly augmented by 2 Gy IR. Addition of KitL only marginally elevated VEGF levels in supernatant of mast cell cultures. VEGF release depended on the presence of MMP-9, because baseline VEGF secretion from MMP-9−/− mast cells was low, and IR only slightly enhanced VEGF release (Fig. S2 I). The observed differential release of VEGF from mast cells with and without IR was not due to changes in mast cell numbers (unpublished data). The stromal cell line MS-5, on the other hand, produced little VEGF, and VEGF release did not change with IR ( D). Using a more complex BM-derived stromal cell population from MMP-9+/+ animals, VEGF secretion did not change before (1961 ± 143 pg/mL) or after IR (2107 ± 150 pg/mL).
Which cell type produces KitL? We show that IR increased KitL release by MS-5 cells in vitro, which could be blocked by a synthetic metalloproteinase inhibitor ( E). In contrast, mast cells only showed baseline secretion of KitL and no change in KitL production after IR. Thus our results indicate cooperation between two cell types in mobilizing mast cells: stromal cells produce KitL, while mast cells produce VEGF in response to IR, and both depend on MMP-9.
To test if IR-induced vasculogenesis/angiogenesis is dependent on mast cells in vivo, mast cell–deficient Sl/Sld mice and WBB6F1+/+ controls received 2 Gy IR after induction of HL ischemia. Necrosis was macroscopically detectable on day 7 in Sl/Sld, but not as profound in the WT mice ( F). By day 14, blood flow recovered in WBB6F1+/+ littermates, whereas Sl/Sld mice showed complete limb amputation (). IR accelerated the ischemic recovery in WBB6F1+/+ mice, but not in Sl/Sld mice ( G). When Sl/Sld mice with HL ischemia were treated with IR, all mice died by day 14. These data support the hypothesis that improved angiogenesis after IR is driven by mast cells.
rKitL protects against lethal IR and increases the number of progenitors (21
). Mobilization of hematopoietic ( A) and endothelial progenitors ( B) following HL ischemia was similar in WBB6F1+/+
mice; however, IR-induced progenitor mobilization was completely blocked in Sl/Sld
but not WBB6F1+/+
mice, indicating that IR-induced progenitor mobilization depends on the presence of KitL. In confirmation of our data that mast cells are the source of VEGF following HL ischemia and IR, we detected lower VEGF plasma levels in Sl/Sld
mice after HL ischemia as compared with WBB6F1+/+
controls ( C); in addition, IR enhanced VEGF plasma levels in WBB6F1+/+
but not Sl/Sld
Figure 5. Irradiation-induced angiogenesis is dictated by MMP-9–mediated factor release from mast and stroma cells and incorporation of BM-derived cells. (A–C) HL ischemia was induced in Sl/Sld and WBB6F1+/+ mice, followed by 2 Gy IR or no IR ( (more ...)
If VEGF signaling following IR promotes angiogenesis under ischemic conditions, administration of neutralizing antibodies against VEGFR-1 and VEGFR-2 should prevent IR-induced angiogenesis. MMP-9+/+ mice received vessel ligation followed by TBI of 2 Gy ( D). Macroscopic examination at day 7 revealed that 2 Gy of IR completely prevented signs of limb necrosis, whereas amputation occurred following HL ischemia ( D, b). The effect of IR in preventing tissue necrosis/amputation was blocked by anti–VEGFR-2 treatment or a combination of anti–VEGFR-1 and anti–VEGFR-2 treatment by day 10, but was only partially inhibited in mice receiving anti–VEGFR-1 by day 14 ( D). Single injections of anti–VEGFR-1 resulted in necrosis but not complete limb amputation.
Administration of rVEGF intraperitoneally caused up-regulation of MMP-9 in BM ( E, a) and increased plasma levels of KitL in treated mice ( E, b). If VEGF promotes mast cell migration into ischemic tissue, blockade of VEGF signaling using VEGFR-1 or VEGFR-2 mAbs should decrease the number of mast cells in the muscle tissue following HL ischemia and/or IR. Indeed, HL ischemia–induced mice treated with VEGFR-2 mAbs or a combination of VEGFR-1 and VEGFR-2 mAbs showed a decreased number of mast cells in the muscle tissue compared with untreated controls (Fig. S2 J). Administration of VEGFR-1 diminished the number of mast cells partially. These data underscore the importance of VEGF signaling in regulating the number of resident mast cells under “stress conditions.”
IR promotes angiogenesis by altering the tissue microenvironment
Local low-dose IR rather than TBI would be a more relevant treatment for future clinical applications by reducing systemic side effects. Therefore, C57BL/6 mice received unilateral HL ischemia. Groups of mice were then administered local IR (2 Gy) of the ischemic limb, IR of the contralateral nonischemic limb, or no IR. IR of the contralateral, nonischemic limb controlled for effects due to BM IR. Muscle tissue regeneration was faster in mice when the ischemic limb was irradiated compared with nonirradiated controls ( F). Local IR of the contralateral, nonischemic limb did not result in faster tissue regeneration. Thus, activation of the BM alone was not sufficient to promote angiogenesis. These data implicate that IR of the ischemic tissue, but not of nonischemic tissue, “conditions” the ischemic tissue microenvironment and is critical for IR-induced angiogenesis.
To understand if progenitors mobilized after IR alone could improve vasculogenesis/angiogenesis, PBMCs isolated from irradiated and unirradiated donor mice were transplanted into groups of C57BL/6 recipient mice after HL ischemia surgery. We observed HL ischemic recovery in mice transplanted with PBMCs from unirradiated mice, but not in mice transplanted with PBMCs isolated from 2 Gy irradiated donors ( G). These data implicated that circulating cells induced by IR alone were not sufficient to duplicate the previously observed angiogenesis-promoting effects of IR.
Do BM-derived cells play any role at all in IR-induced angiogenesis under ischemic conditions? When C57BL/6 mice reconstituted with donor GFP-expressing BM cells received HL surgery and 2 Gy IR, BM-derived GFP+ cells contributed to vessel formation in muscle tissue of mice that had undergone HL ischemic surgery ( I). These BM-derived cells were covered by smooth muscle actin (SMA)+ cells, indicating their incorporation into mature vessels. The highest density of donor-derived GFP+ vessels was found in animals that had received HL ischemic surgery and 2 Gy IR (82%) ( I). Donor-derived GFP+ cells were undetectable in unirradiated mice without HL ischemia (0%). These data imply that IR applied under ischemic conditions promotes incorporation of BM-derived cells into regenerating vasculature.
To understand whether IR conditions the local ischemic microenvironment to promote mast cell incorporation, we induced bilateral HL ischemia in WBB6F1+/+ mice. IR of one leg resulted in faster recovery of blood flow compared with the unirradiated ischemic limb ( H). Increased numbers of mast cells were found in muscle tissue of the irradiated but not the nonirradiated limb (Fig. S2 K, a and b). Plasma levels for VEGF and KitL after bilateral HL ischemia further increased following loco-regional IR (Fig. S2, K, c).