Matrix Metalloproteinase-9 Is Essential for Ischemia-Induced Neovascularization by Modulating Bone Marrow–Derived Endothelial Progenitor Cells
Objective— Both matrix metalloproteinases (MMPs) and endothelial progenitor cells (EPCs) have been implicated in the process of neovascularization. Here we show that the impaired neovascularization in mice lacking MMP-9 is related to a defect in EPC functions in vasculogenesis.
Methods and Results— Hindlimb ischemia surgery was conducted in MMP-9−/− mice and wild-type (MMP-9+/+) mice. Blood flow recovery was markedly impaired in MMP-9−/− mice when compared with that in wild-type mice as determined by laser Doppler imaging. Flow cytometry demonstrated that the number of EPC-like cells (Sca-1+/Flk-1+) in peripheral blood increased in wild-type mice after hindlimb ischemia surgery and exogenous vascular endothelial growth factor stimulation, but not in MMP-9−/− mice. Plasma levels and bone marrow concentrations of soluble Kit-ligand (sKitL) were significantly elevated in wild-type mice in response to tissue ischemia, but not in MMP-9−/− mice. C-kit positive bone marrow cells of MMP-9−/− mice have attenuated adhesion and migration than those isolated from wild-type mice. In in vitro studies, incubation with selective MMP-9 inhibitor suppressed the colony formation, migration, and tube formation capacities of EPC. Transplantation of bone marrow cells from wild-type mice restored collateral flow formation in MMP-9−/− mice.
Conclusions— These findings suggest that MMP-9 deficiency impairs ischemia-induced neovascularization, and these effects may occur through a reduction in releasing the stem cell-active cytokine, and EPC mobilization, migration, and vasculogenesis functions.
Angiogenesis, defined as sprouting of new blood vessels from preexisting vascular structures, is a process necessary for wound healing and is a physiological response to tissue ischemia.1 In recent years, our understanding of the process responsible for new vessel formation after tissue ischemia has been changing. Increasing evidence suggests that this process may start with degradation of nonfibrillar collagens in basement membrane, followed by migration and proliferation of preexisting, fully differentiated vascular endothelial cells, and more importantly, incorporation and differentiation of circulating endothelial progenitor cells (EPCs) into endothelial cells in situ.2–5 These circulating EPCs, originally derived from bone marrow, could be mobilized endogenously as triggered by tissue ischemia or exogenously by cytokine stimulation.4,5 Enhanced mobilization of EPCs augments the neovascularization of ischemic tissue and may be clinically relevant in the setting of tissue ischemia.6–8 Currently, 2 types of EPCs, namely early and late EPCs, can be isolated and identified from peripheral blood,9,10 but the detailed regulation of individual matrix metalloproteinases (MMPs) in EPCs, especially late EPCs for vasculogenesis, has not been clarified.
The process of new vessel formation is thought to occur mainly through capillary splitting, also know as intussusception or capillary budding, which leads to branching and is associated with extracellular matrix remodeling and degradation of the vascular basement membrane to allow endothelial cells to migrate and invade into the surrounding tissue.11,12 MMPs are a family of zinc-dependent extracellular proteinases comprising at least 20 members that are collectively capable of degrading all known extracellular matrix components. MMP-2 and MMP-9, which belong to the gelatinase subclass of the MMP family, have been shown to be upregulated after tissue ischemia and have been suggested to initiate angiogenesis by degrading nonfibrillar collagens in response to hypoxia.13–15 It was also shown that, although upregulated in angiogenic lesions, MMP-2 and MMP-9 can promote the release of extracellular matrix-bound cytokines, such as vascular endothelial growth factor (VEGF), to regulate angiogenesis.16 However, the absence of MMP-2 does not impair the induction of angiogenesis during the carcinogenesis of pancreatic islets, which suggests that MMP-9 but not MMP-2 is a more important component of the angiogenic switch.16 It is thus important to define whether and how MMP-9 could directly contribute to neovascularization in response to different pathophysiological conditions, such as tissue ischemia. Considering the pivotal role of circulating EPCs for vasculogenesis, we hypothesize that the expression of MMP-9 may not only contribute to matrix degradation but may also directly modulate the behaviors and functions of circulating EPCs responding to tissue ischemia. Furthermore, MMP-9 may play the cardinal role in in vivo neovascularization through its direct effect on bone marrow–derived circulating EPCs. Accordingly, in this study, we show the influence of the targeted deletion of the MMP-9 gene on ischemia-induced neovascularization and address the potential mechanistic link between MMP-9 and EPCs in response to tissue ischemia.
Materials and Methods
We performed the following in vivo: ischemic hindlimb perfusion assay, histological analysis (capillary density, analysis of collateral vessel formation, analysis of EPCs, bone marrow cell transplantation), biological analysis (gelatin zymography and immunoassay). Ex vivo, we performed aortic-ring culture assay; in vitro, cell isolation (human early and late EPC, mouse bone marrow–derived c-Kit+ cells), colony-forming assay, cell adhesion assay, senescence assay, cell migration and invasion assays, cell proliferation assay, and immunocytofluorescence as detailed in the supplemental materials (available online at http://atvb.ahajournals.org).
Quantitative data are expressed as means±SEM. Statistical analysis was adequately performed by the unpaired Student t test or analysis of variance followed by Scheffe multiple-comparison post hoc test. Data were analyzed using SPSS software (version 14; SPSS). A probability value of <0.05 was considered to indicate statistical significance.
MMP-9 Deficiency Impairs Ischemia-Induced Angiogenesis
To evaluate the role of MMP-9 in ischemia-induced angiogenesis, we induced unilateral hindlimb ischemia in MMP-9−/− mice and background-matched wild-type mice (n=8 per group). The blood flow of the ischemic and nonischemic legs was monitored weekly by laser Doppler imaging (Figure 1A). In wild-type mice, the blood flow of the ischemic leg recovered gradually, reaching approximately 90% of the blood flow of the untreated leg by 5 weeks, but blood flow recovery was significantly impaired in MMP-9−/− mice (P<0.05; Figure 1B). In addition, histological analysis revealed that the capillary density in the ischemic limb was significantly increased in MMP-9+/+ mice, whereas no such increase was noted in MMP-9−/− mice (capillary/myofiber ratio: 1.2±0.1 versus 0.6±0.2/mm2, P<0.001; Figure 1C). Half of the MMP-9−/− mice suffered from spontaneous foot amputation, but the limbs were preserved in all of the MMP-9+/+ mice (Figure 1D). These data indicate that new vessel formation and blood flow recovery were impaired in MMP-9−/− mice exposed to tissue ischemia.
Effects of Acute Ischemia on Expression and Activity of MMP-2, MMP-9, Tissue Inhibitors of Metalloproteinase-1, and Soluble Form of Kit-Ligand
Gelatin zymographic analysis showed that MMP-2 significantly upregulated in the ischemic muscle of wild-type and MMP-9−/− mice at day 3 after ligation of femoral artery (n=6 for each group). As expected, no MMP-9 activity was detected in the ischemic tissue of MMP-9-deficient mice, but MMP-9 significantly activated in wild-type mice after hindlimb ischemia surgery (Figure 2A). As shown in Figure 2B and 2C, levels of MMP-9 but not MMP-2 significantly elevated in bone marrow and in peripheral blood in wild-type mice at day 3 after tissue ischemia. Plasma and bone marrow levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) were significantly increased in wild-type mice, but the activation of TIMP-1 was not observed in bone marrow tissue in MMP-9−/− mice (Figure 2D). Furthermore, impaired releasing of soluble form of Kit-ligand (sKitL) was noted in MMP-9–deficient mice in bone marrow and in peripheral blood (Figure 2E), suggesting a critical role for MMP-9 in rapidly releasing the stem cell-active cytokine.
Deficiency of MMP-9 Reduces EPC-Like Cell Mobilization
To investigate EPC-like cell mobilization after tissue ischemia and exogenous VEGF stimulation in wild-type mice and MMP-9−/− mice, levels of Sca-1+/Flk-1+ cells in peripheral blood were determined by flow cytometry. The basal number of EPC-like cells did not differ significantly between wild-type mice (0.58±0.11%) and MMP-9−/− mice (0.68±0.09%; P=0.502; n=6 for each group, Figure 2F). As shown in previous studies,4,22 mobilization of EPCs contributed to postnatal neovascularization and was enhanced by tissue ischemia or cytokine administration in wild-type mice (baseline versus ischemia surgery versus VEGF stimulation, 0.58±0.11 versus 1.65±0.19 versus 1.80±0.20%, P=0.001). However, levels of Sca-1+/Flk-1+ cells in peripheral blood were not significantly elevated in response to acute ischemia or VEGF stimulation in MMP-9−/− mice (0.68±0.09 versus 0.70±0.19 versus 1.23±0.18%, P=0.064).
Characterization of Human EPCs
Early and late EPCs were isolated from peripheral blood MNCs of healthy subjects as previously described.10 The peripheral blood MNCs that initially seeded on fibronectin-coated wells were round in shape (supplement Figure IA). After the medium was changed on day 4, attached early EPCs appeared to be elongated with a spindle shape (supplement Figure IB). Late EPCs with a cobblestone-like morphology similar to mature endothelial cells were grown to confluence (supplement Figure IC). A colony-forming unit (CFU) of EPCs was defined as a central core of round cells with elongated sprouting cells at the periphery (supplement Figure ID). EPC colony was further confirmed as cells double positive for acLDL uptake (supplement Figure IE) and lectin (UEA-1) binding affinity (supplement Figure IF). Late EPC characterization was performed by immunohistochemical staining, and most of the cells expressed mature endothelial markers, VE-cadherin (supplement Figure IG), PECAM-1 (CD31; supplement Figure IH), and CD34 (supplement Figure II), which are considered critical markers of late EPCs.
Effects of Selective MMP-9 Inhibitor on EPC Number, Proliferation, and Colony-Forming Capacity
After seeding MNCs on 6-well plates, cells were incubated with different concentrations of selective MMP-9 inhibitor (0.01 to 1.00 μmol/L) for 4 days. Compared with that in the control group, incubation of MNCs with MMP-9 inhibitor did not decrease the amount of differentiated, adherent, early EPCs assessed by fluorescein isothiocyanate lectin and DiI-acLDL staining (control versus 0.01 versus 0.10 versus 1.00 μmol/L, 140±8 versus 132±11 versus 127±13 versus 131±4/HPF, P=0.819). The effect of MMP-9 inhibitor on late EPC proliferation was analyzed by MTT assay. Incubation of late EPCs with different concentrations of MMP-9 inhibitor did not significantly affect EPC proliferation activity (1.00±0.02 versus 1.14±0.03 versus 1.16±0.03 versus 1.14±0.08; P=0.681). However, the number of EPC CFUs formed after 5 days of cell culture was suppressed after treatment with MMP-9 inhibitor (57±4 versus 42±4 versus 37±3 versus 35±3 CFU/well; P<0.05 versus control, Figure 3A), which suggests that the colony formation of EPCs requires MMP-9.
Effects of MMP-9 Inhibitor on EPC Senescence, Migration, and Tube Formation
To determinate the onset of cellular aging, acidic β-galactosidase was detected as a biochemical marker for acidification typical of EPC senescence. Compared with the control group, incubation of either early or late EPCs with MMP-9 inhibitor (0.01 to 1.00 μmol/L) for 4 days did not significantly enhance the percentage of senescence-associated β-galactosidase-positive EPCs (early EPCs, P=0.732; late EPCs, P=0.850), which suggests that suppression of MMP-9 activity did not promote either early or late EPC aging.
A modified Boyden chamber assay using VEGF as a chemoattractic factor was performed to evaluate the effect of selective MMP-9 inhibitor on the migratory capacity of EPCs. After 4 days of culturing, MMP-9 inhibitor dose-dependently suppressed the VEGF-induced migration of late EPCs (control versus 0.01 versus 0.10 versus 1.00 μmol/L, 61±3 versus 50±5 versus 26±1 versus 20±3/HPF; *P<0.05, **P<0.01 compared with control group, Figure 3B), which suggests VEGF-induced EPC migration is MMP-9–dependent.
An in vitro angiogenesis assay was performed with late EPCs to investigate the effect of MMP-9 inhibition on EPC neovascularization. After 4 days of culturing, the functional capacity for tube formation of late EPCs on ECMatrix gel was significantly attenuated in the MMP-9 inhibitor-treated group compared with the control group (P<0.05 versus control; Figure 3C).
MMP-9 Deficiency Impairs Ex Vivo Angiogenesis in Mouse Aortic-Ring Culture Assay
As shown in Figure 3D, wild-type and MMP-9−/− mouse aortic rings in endothelial basal medium (serum-free EBM)-2 without VEGF mounted a weak tubulogenic response. Neither MMP-2 nor MMP-9 activity could be detected in serum-free EBM-2 medium by gelatin zymographic analysis (data not shown). Administration of VEGF (20 ng/mL) significantly enhanced the tubulogenic response in aortic rings from wild-type and MMP-9−/−. However, quantitative analysis revealed that the mean density of the microvessels was significantly higher in the wild-type aorta than in the MMP-9−/− aorta (P<0.05, Figure 3D). Treatment with recombinant MMP-9 (rMMP-9, 0.5 μg/mL) in the presence of VEGF markedly enhanced its angiogenic response in the aorta rings of wild-type and MMP-9−/− than VEGF alone (compared with VEGF alone, both P<0.005). These data provided ex vivo evidence that deficiency of MMP-9 activity impairs microvessel formation and the vasculogenesis ability of EPCs.
Lack of MMP-9 Attenuates C-Kit–Positive Bone Marrow Cell Adhesion and Migration
C-kit–positive bone marrow cells were purified and investigated the role of MMP-9 in cell functions. As shown in Figure 3E, c-kit–positive bone marrow cells isolated from MMP-9−/− mice were demonstrated have attenuated adhesion (100±11 versus 73±3, P=0.011) and migration capacities (11±6 versus 5±4/HPF, P=0.006), suggesting a critical role of MMP-9 in functions of progenitor cells in bone marrow.
Wild-Type Mice Bone Marrow Rescues Neovascularization in MMP-9−/− Mice
As presented in Figure 4A, collateral flow recovery after hindlimb surgery was significantly augmented in MMP-9−/− mice receiving wild-type mouse (eGFP transgenic mice) bone marrow cells compared with that in MMP-9−/− mice receiving MMP-9−/− mouse bone marrow cells, which indicates that restoration of MMP-9–deficient mice with wild-type mouse bone marrow cells could rescue impaired neovascularization in ischemic tissue. Furthermore, significantly impaired neovascularization after hindlimb surgery was noted in wild-type mice receiving MMP-9−/− mouse bone marrow cells compared with that in wild-type mice receiving wild-type mouse bone marrow cells, which suggests a critical role of MMP-9 in bone marrow cells in the process of neovascularization. By immunofluorescence staining, more GFP+/CD31+ double-positive cells were identified in wild-type mice than in MMP-9−/− mice, both of which received eGFP mouse bone marrow cells (36±4 versus 13±2/HPF, P<0.001; Figure 4B).
The current results suggest that MMP-9 deficiency may impair ischemia-induced neovascularization, which could be through a reduction in EPC mobilization, migration, and functions in vasculogenesis. These findings gave further support to previous studies suggesting that MMP-9 and bone marrow–derived EPCs play a pivotal role in neovascularization in response to tissue ischemia,5,24 and provide novel evidence in explaining the comprehensive and multi-functional role of MMP-9.
Recently, it has become more clear that the role of MMPs in angiogenesis is more complex than simply degrading the extracellular matrix to facilitate invasion of endothelial cells.25 As mentioned previously, MMP-2 and MMP-9 have been shown to play an important role in initiating angiogenesis,16 and were upregulated after tissue ischemia,16,24,26 and could promote the release of extracellular matrix-bound cytokines, such as VEGF, which can regulate angiogenesis. In recent study, Cheng and colleagues indicated that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cells and EPC invasion or proliferation and mobilization of EPCs.26 Reduced MMP-9 expression has been shown to be associated with impaired circulating progenitor cell migration and invasion in the case of hyperglycemia.27 However, recent in vivo data suggest that the absence of MMP-2 did not impair the induction of angiogenesis during the carcinogenesis of pancreatic islets.16 This finding led to the hypothesis that MMP-9, rather than MMP-2, plays the critical role in angiogenic switch. In the present study, we used a gene-targeting strategy to demonstrate that MMP-9 plays a critical role in ischemia-induced neovascularization, and showed that not only MMP-2, but also MMP-9, can modulate vasculogenesis activities of EPCs through a reduction of mobilization, migration, and angiogenesis functions of EPCs.
Improved neovascularization in response to tissue ischemia is an important therapeutic strategy to reduce organ damage. Convincing evidence suggests that neovascularization in adults is not solely the result of the proliferation of endothelial cells (angiogenesis) but also involves circulating EPCs in the process of vasculogenesis.3–5 These circulating EPCs are derived from bone marrow and are mobilized endogenously, triggered by tissue ischemia, or exogenously by cytokine stimulation, such as VEGF and stromal cell-derived factor-1 (SDF-1).4,5 A recent study using a mouse bone marrow suppression model indicated that cytokine-induced progenitor cell mobilization is dependent on the local secretion of MMP-9 by the hematopoietic and stromal compartments of the bone marrow, which results in the release of sKitL (also known as stem cell factor).25 Using a mouse hindlimb ischemia model, our data demonstrated that lack of MMP-9 impaired releasing of sKitL in bone marrow, implying a critical role for MMP-9 in rapidly releasing the stem cell–active cytokine. As shown in previous studies,4,22 mobilization of EPCs contributed to postnatal neovascularization mobilization of bone marrow-derived EPCs to circulation occurs in response to tissue ischemia or exogenous EVGF stimulation, and this process is MMP-9-dependent. It is also interesting to find that markedly impaired neovascularization after hindlimb surgery in wild-type mice receiving MMP-9−/− mice bone marrow cells, which suggests the critical role of MMP-9 in bone marrow cells in ischemia-induced neovascularization. Additionally, bone marrow–derived EPCs differentiated into endothelial cells (defined as GFP+CD31+ cells) were significantly decreased in ischemic tissue around the vessels in MMP-9−/− mice than in wild-type mice, which implies that MMP-9 activation in ischemic tissue is required in the homing process of EPCs. These findings provide a new treatment strategy to attenuate organ damage in the acute ischemic stage by promoting MMP-9 activity in bone marrow cells or in ischemic tissue. Furthermore, Ahn and coworkers reported that MMP-9 is required for vasculogenesis but not for angiogenesis in tumor growth.28 However, CD11b-positive myelomonocytic cells but not bone marrow–derived EPCs were mainly responsible for the development of immature blood vessels in MMP-9–deficient mice receiving wild-type bone marrow cells. These findings suggests that diverse types of bone marrow cells or bone marrow–derived EPCs can be mobilized to circulation and contribute to vasculogenesis in response to tissue ischemia or tumor growth, but MMP-9 was found to play a critical role in the process.
The migratory function of EPCs in response to VEGF plays a critical role during neovascularization.22 In an in vitro study, we observed that MMP-9 inhibitor directly suppressed VEGF-induced EPC migration, which suggests that matrix degradation by MMP-9 may directly facilitate the migration of EPCs, and also exposed cryptic sites enhancing angiogenesis or release matrix-bound proangiogenic growth factors. Our data further support the notion that the recovery of MMP-9 activity in bone marrow cells can further promote bone marrow–derived EPCs to infiltrate in ischemic tissues and differentiate into endothelial cells, which was shown in MMP-9−/− mice with transplanted eGFP mice bone marrow.
In addition, recent studies have suggested that at least 2 different types of EPCs, early and late EPCs, could be identified in an ex vivo culture system.9,10,23 However, little data had been provided on the individual effect of MMP-9 on different types of EPCs. In this ex vivo study, we for the first time demonstrated the detrimental effect of MMP-9 inhibition on the migration and vasculogenesis activities of late EPCs, which implies a multifunctional role of MMP-9 in ischemia-induced neovascularization. Additionally, MMP-9 inhibition had no effect on EPC proliferation and senescence in vitro, suggesting that MMP-9 may contribute to vasculogenesis specifically by promoting the invasive and migratory activities of EPCs. It is also interesting to find that MMP-9 inhibition suppressed the EPC colony formation, but had no effect on early EPC numbers and late EPC proliferation, implying that colony formation by EPCs is not only related to EPC numbers, but may also reflect EPC vasculogenesis function. Our findings on EPCs may be of particular importance in clinical implication because EPCs possess the vasculogenesis capacity and may serve as a potential therapeutic target for vascular regeneration in ischemic tissue.
Our findings indicate that MMP-9 deficiency can impair ischemia-induced neovascularization, which may occur through a reduction in EPC mobilization and vasculogenesis functions. This direct biological function of MMP-9 on EPCs provides a novel mechanism in addition to the current understanding about MMP-9 in ischemia-induced neovasculogenesis and may be exploited for the potential therapeutic target of neovascularization in clinical tissue ischemia.
Sources of Funding
This study was partly supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology and the Ministry of Health, Labor, and Welfare of Japan; NSC 95-2314-B-010-016 and 96-2320-B-039-042 from the National Science Council, Taiwan; VGH-96DHA0100478, VGH-97DHA0100127, and VGH-ER-2-97DHA0100664 from Taipei Veterans General Hospital, Taipei, Taiwan; CMU95-288, CMU96-188, and CMU96-052 from China Medical University; CI 96-16 from the Yen Tjing Ling Medical Foundation, Taipei, Taiwan; and a grant from the Ministry of Education, Aim for the Top University Plan.
Y.-H.C. and C.-H.W. contributed equally to this study.
Received August 27, 2008; revision accepted April 27, 2009.
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