Angiogenesis, defined as sprouting of new blood vessels

Transcription

1 Matrix Metalloproteinase-9 Is Essential for Ischemia-Induced Neovascularization by Modulating Bone Marrow Derived Endothelial Progenitor Cells Po-Hsun Huang, Yung-Hsiang Chen, Chao-Hung Wang, Jia-Shiong Chen, Hsiao-Ya Tsai, Feng-Yen Lin, Wei-Yuh Lo, Tao-Cheng Wu, Masataka Sata, Jaw-Wen Chen, Shing-Jong Lin Downloaded from by guest on July 21, 2018 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. (Arterioscler Thromb Vasc Biol. 2009;29: ) Key Words: matrix metalloproteinases endothelial progenitor cells neovascularization 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 Received August 27, 2008; revision accepted April 27, From the Division of Cardiology, Department of Internal Medicine (P.-H.H., T.-C.W., J.-W.C., S.-J.L.), Taipei Veterans General Hospital; the Institute of Clinical Medicine (P.-H.H., H.-Y.T., S.-J.L.); and the Cardiovascular Research Center (P.-H.H., W.-Y.L., T.-C.W., J.-W.C., S.-J.L.), National Yang-Ming University, Taipei, Taiwan; the Department of Cardiovascular Medicine (P.-H.H., M.S.), University of Tokyo Graduate School of Medicine, Tokyo, Japan; the Graduate Institute of Integrated Medicine (Y.-H.C.), College of Chinese Medicine, China Medical University, Taichung, Taiwan; the Division of Cardiology, Department of Internal Medicine (C.-H.W.), Chang Gung Memorial Hospital, Keelung; Chang Gung University College of Medicine, Taoyuan, Taiwan; Institute and Department of Pharmacology (J.-S.C., J.-W.C.), National Yang-Ming University; and the Division of Anesthesiology (F.-Y.L.), Taipei Medical University Hospital and Department of Anesthesiology, School of Medicine (F.-Y.L.), Taipei Medical University, Taipei, Taiwan. Y.-H.C. and C.-H.W. contributed equally to this study. Correspondence to Shing-Jong Lin, MD, PhD, the Division of Cardiology, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan. sjlin@vghtpe.gov.tw or jwchen@vghtpe.gov.tw or msata-circ@umin.net 2009 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 1180 Arterioscler Thromb Vasc Biol August 2009 Downloaded from by guest on July 21, 2018 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 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 Statistical Analysis 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. Results 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 Figure 1. Reduced collateral flow formation and capillary density in MMP-9 deficient mice. Blood flow recovery was markedly impaired in MMP-9 / mice compared with that in wildtype mice as determined by laser Doppler imaging (n 8 for each group; A and B). Anti-CD31 immunostaining showed less developed collateral vessels and capillary formation in MMP- 9 / mice (C). Some MMP-9 deficient mice suffered from spontaneous foot amputation, but none of the wild-type mice lost their limbs (D). (*P 0.05 compared with wild-type mice.) 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: versus /mm 2, 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

3 Huang et al MMP-9 and Ischemia-Induced Neovascularization 1181 Downloaded from by guest on July 21, 2018 Figure 2. Effects of acute ischemia on expression of MMPs, TIMP-1, skitl, and EPC-like cell mobilization. Zymographic detection of MMP-2 and MMP-9 gelatinolytic activities in ischemic muscle were assessed from wild-type and MMP-9 / mice (n 6 for each group; A). (*P 0.05 compared with wild-type mice.) Plasma and bone marrow concentrations of MMP-2 (B), MMP-9 (C), TIMP-1 (D), and skitl (E) were determined at baseline and at day 3 after hindlimb ischemia surgery by ELISA. EPC-like cell mobilization (defined as Sca-1 /Flk-1 cells) after tissue ischemia and exogenous VEGF stimulation was determined by flow cytometry (F). (*P 0.05 vs baseline). 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 wildtype mice ( %) and MMP-9 / mice ( %; 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, versus versus %, 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 ( versus versus %, 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 fibronectincoated 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 Figure 3. The role of MMP-9 in EPC functions, capillary microvessel sprouting, and bone marrow progenitor cell functions. The number of EPC colony-forming units was suppressed by MMP-9 inhibitor (A). A modified Boyden chamber assay and in vitro angiogenesis assay were used to evaluate the effect of MMP-9 inhibitor on EPC migration (B) and neovascularization (C). Photomicrographs of an aortic-ring cultured serum-free EBM-2 medium with or without VEGF and recombinant MMP-9 for 2 weeks, respectively, showing microvessel sprouting circumscribing a patent lumen (D). C-kit positive bone marrow cells from wild-type and MMP-9 / mice were tested for cell functions (E). 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 mol/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 mol/l, versus versus 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 ( versus versus versus ; 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.

4 1182 Arterioscler Thromb Vasc Biol August 2009 Downloaded from by guest on July 21, 2018 Effects of MMP-9 Inhibitor on EPC Senescence, Migration, and Tube Formation To determinate the onset of cellular aging, acidic -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 mol/l) for 4 days did not significantly enhance the percentage of senescence-associated -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 dosedependently suppressed the VEGF-induced migration of late EPCs (control versus 0.01 versus 0.10 versus 1.00 mol/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 Figure 4. Wild-type mice bone marrow rescues neovascularization in MMP-9 / mice. Collateral flow formation was evaluated 2 weeks after hindlimb ischemia by laser Doppler imaging system (A; **P 0.05 compared with that in MMP-9 / mice receiving GFP mouse bone marrow cells; *P 0.05 compared with WT mice receiving GFP mouse bone marrow cells). By immunofluorescence staining, more GFP /CD31 double-positive cells (yellow color, blue arrowheads) were identified in wild-type mice than in MMP-9 / mice, both of which received egfp mouse bone marrow cells (B; *P 0.05 compared with WT mice receiving GFP mouse bone marrow cells). 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 g/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 ( 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 wildtype 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

5 Huang et al MMP-9 and Ischemia-Induced Neovascularization 1183 Downloaded from by guest on July 21, 2018 egfp mouse bone marrow cells (36 4 versus 13 2/HPF, P 0.001; Figure 4B). Discussion 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 multifunctional 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, CD11bpositive 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.

6 1184 Arterioscler Thromb Vasc Biol August 2009 Downloaded from by guest on July 21, 2018 Conclusions 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 B and B from the National Science Council, Taiwan; VGH-96DHA , VGH-97DHA , and VGH- ER-2-97DHA from Taipei Veterans General Hospital, Taipei, Taiwan; CMU95-288, CMU96-188, and CMU from China Medical University; CI from the Yen Tjing Ling Medical Foundation, Taipei, Taiwan; and a grant from the Ministry of Education, Aim for the Top University Plan. None. Disclosures References 1. Folkma J. Clinical applications of research on angiogenesis. N Engl J Med. 1995;333: Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1: Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. 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7 Downloaded from by guest on July 21, 2018 Matrix Metalloproteinase-9 Is Essential for Ischemia-Induced Neovascularization by Modulating Bone Marrow Derived Endothelial Progenitor Cells Po-Hsun Huang, Yung-Hsiang Chen, Chao-Hung Wang, Jia-Shiong Chen, Hsiao-Ya Tsai, Feng-Yen Lin, Wei-Yuh Lo, Tao-Cheng Wu, Masataka Sata, Jaw-Wen Chen and Shing-Jong Lin Arterioscler Thromb Vasc Biol. 2009;29: ; originally published online May 21, 2009; doi: /ATVBAHA Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2009 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at:

8 Supplement material Matrix metalloproteinase-9 is essential for ischemia-induced neovascularization by modulating bone marrow-derived endothelial progenitor cells Po-Hsun Huang, Yung-Hsiang Chen, Chao-Hung Wang, Jia-Shiong Chen, Hsiao-Ya Tsai, Feng-Yen Lin, Wei-Yuh Lo, Tao-Cheng Wu, Masataka Sata, Jaw-Wen Chen, Shing-Jong Lin Materials and methods Animals All mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and all mice were kept in microisolator cages on a 12-h day/night cycle. All experimental procedures and protocols involving animals were in accordance with the local institutional guidelines for animal care of the University of Tokyo (Tokyo, Japan), and approved by the institutional animal care committee of National Yang-Ming University (Taipei, Taiwan), and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No , revised 1985). Mouse Hindlimb Ischemic Model In the present study, we used 8-week-old male wild-type mice (MMP-9 +/+ mice) and MMP-9 -/- mice of FVB background. After 2-week stabilization period, unilateral hindlimb ischemia was induced by excising the right femoral artery as previously described. 17 Briefly, the animals were anesthetized by 1

9 intraperitoneal injection of pentobarbital (50 mg/kg). The proximal and distal portions of the right femoral artery and the distal portion of the right saphenous artery were ligated. After that, the arteries and all side branches were dissected free and excised. Hindlimb blood perfusion was measured with a laser Doppler perfusion imager (LDPI) system (Moor Instruments Limited, Devon, UK) before and after the surgery and was then followed up weekly. Excess hairs were removed from the limbs by using depilatory cream before imaging, and the mice were placed on a heating plate at 40 C. To avoid the influence of ambient light and temperature, the results were expressed as the ratio of perfusion in the right (ischemic) versus left (nonischemic) limb. Measurement of Capillary Density in the Ischemic Leg Five weeks after surgery, the mice were sacrificed by intraperitoneal injection of an overdose of pentobarbital. The whole limbs were fixed in methanol overnight. The femora were carefully removed, and the ischemic thigh muscles were embedded in paraffin. Sections (5 μm) were de-paraffinized and incubated with a rat-monoclonal antibody against murine CD31 (clone MEC13.1, BD PharMingen, San Diego, CA, USA). Antibody distribution was visualized with the use of the avidin-biotin-complex technique and Vector Red chromogenic substrate (Vector Laboratories, Burlingame, CA, USA), followed 2

10 by counterstaining with hematoxylin. Capillaries were identified by positive staining for CD31 and morphology. Three cross-sections were analyzed for each animal and ten different fields from each tissue preparation were randomly selected, and visible capillaries were counted. Capillary density was expressed as the number of capillaries per square millimeter. Gelatin Zymography and Immunoassay for MMP-2, MMP-9, Tissue Inhibitors of Metalloproteinase-1 (TIMP-1) and soluble form of Kit-ligand (skitl) MMP-9 and MMP-2 activities in ischemia muscle were determined using SDS-PAGE gelatin zymographic analysis, using 20 μg protein per sample (n=6 for each group). The samples contained both latent and activated forms, revealed in the presence of SDS. After incubation, zynographies were imaged and quantified using GelDoc 1000 (Biorad).Gelatinolytic activities were separately normalized in regards to the standard loaded in each gel. Plasma and bone marrow concentrations of MMP-2, MMP-9, TIMP-1, and skitl were measured using commercial available ELISA (R&D System). All the procedures were carried out according to the instructions of the manufacturers. 3

11 Each standard and each plasma sample were analyzed two times, and the mean values were used for all subsequent data analysis. Mobilization of EPCs in Wild-Type Mice and MMP-9 -/- Mice To examine the role of MMP-9 in EPC mobilization in response to tissue ischemia (48 hours after surgery) and exogenous VEGF (human VEGF, 500 μg/kg/day for 3 days) stimulation, we used fluorescein isothiocyanate (FITC) anti-mouse Sca-1 (ebioscience, San Diego, CA, USA) and phycoerythrin (PE) anti-mouse Flk-1 (VEGFR-2, ebioscience) antibodies. The number of Sca-1 + /Flk-1 + cells in peripheral blood mononuclear cells (MNCs) was examined by fluorescence-activated cell sorter (FACS calibur; Becton Dickinson, San Jose, CA, USA). Circulating EPCs were quantified by enumerating Sca-1 + /Flk-1 + cells (n=6 for each group). Human EPC Isolation and Cultivation Total MNCs were isolated from 20 ml peripheral blood collected from healthy young human volunteers by density gradient centrifugation with Histopaque-1077 (1.077 g/ml; Sigma, St. Louis, MO, USA) as previously described. 10 Briefly, MNCs ( ) were plated in 2 ml endothelial growth medium (EGM-2 MV; Cambrex, East Rutherford, NJ, USA), with supplements 4

12 (hydrocortisone, R 3 -insulin-like growth factor 1, human endothelial growth factor, VEGF, human fibroblast growth factor, gentamicin, amphotericin B, vitamin C, and 20% fetal bovine serum) on fibronectin-coated 6-well plates. Under daily observation, after 4 days of culturing, medium was changed and nonadherent cells were removed; attached early EPCs appeared to be elongated with a spindle shape. Culture medium was replaced every 4 days, and each colony/cluster was followed up. A certain number of early EPCs could continue to grow into colonies of late EPCs, which emerged 2-4 weeks after the start of MNC culture. The late EPCs exhibited a cobblestone morphology and a monolayer growth pattern typical of mature endothelial cells at confluence. 10 Human EPC Colony-Forming Assay Isolated MNCs were resuspended in growth medium (EndoCult TM liquid medium, StemCell Technologies, Vancouver, Canada), and, in total, MNCs were preplated in a fibronectin-coated 6-well plate in duplicate, as previously described. 18 After 48 hours, the nonadherent cells were collected by pipetting the medium in each well up and down 3 times, and cells were replated onto a fibronectin-coated 24-well plate. Cultured cells were then treated with various concentrations of selective MMP-9 inhibitor (selective 5

13 MMP-9 Inhibitor, IC 50 = 5 nm; Calbiochem, San Diego, CA, USA) for 72 hours. On day 5 of the assay, the number of colony-forming units (CFUs) per well for each sample was counted manually in a minimum of 3 wells. EPC Characterization The early EPCs were characterized as adherent cells double positive for acetylated low-density lipoprotein uptake and lectin binding by direct fluorescent staining as previously described. 10 Briefly, the adherent cells were first incubated with 2.4 μg/ml 1,1 -dioctadecyl-3,3,3,3 -tetramethylindocarbocyanine perchlorate-acetylated low-density lipoprotein (DiI-acLDL; Molecular Probe) for 1 hour and then fixed in 2% paraformaldehyde and counterstained with 10 μg/ml FITC-labeled lectin from Ulex europaeus (UEA-1) (Sigma). The late EPC-derived outgrowth endothelial cell population was also characterized by immunofluorescence staining for the expression of VE-cadherin, platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD-31), and CD34 (Santa Cruz). The fluorescent images were recorded under a laser scanning confocal microscope. EPCs Numbers and Proliferation Assay The number of early EPCs and the proliferation of late EPCs were determined by direct staining of 6 random high-power microscopic fields ( 100) and by 6

14 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide (MTT) assay, respectively. 10 Early EPCs were cultured in plates with various concentrations of selective MMP-9 inhibitor. After being cultured for 4 days, the early EPCs were counted and the late EPCs were supplemented with MTT (0.5 mg/ml; Sigma) and incubated for 4 hours for the proliferation assay. The blue formazen was dissolved with dimethyl sulfoxide and measured at 550/650 nm. EPC Senescence Assay Cellular aging was determined with a Senescence Cell Staining kit (Sigma). Briefly, after being washed with phosphate-buffered saline (PBS), both early and late EPCs were fixed for 6 minutes in 2% formaldehyde and 0.2% glutaraldehyde in PBS and then incubated for 12 h at 37 C without CO 2 with fresh X-gal staining solution (1 mg/ml, 5 mmol/l potassium ferrocyanide, 5 mmol/l potassium ferricyanide, and 2 mmol/l MgCl 2 ; ph 6). After being stained, the blue-stained cells and total cells were counted, and the percentage of β-galactosidase-positive cells was calculated. 19 EPC Fibronectin Adhesion Assay Early and late EPCs cultured from healthy volunteers were washed with PBS and gently detached with 0.5 mmol/l EDTA in PBS. After centrifugation and resuspension in basal medium with 5% fetal bovine serum, identical cells 7

15 ( cells per well) were placed on fibronectin-coated 6-well plates and incubated with selective MMP-9 inhibitor for 30 minutes at 37ºC. The cells were gently washed with PBS 3 times after 30 minutes of adhesion, and the adherent cells were counted by independent blinded investigators. Endothelial characteristics of the adherent cells were phenotyped by indirect immunostaining with the use of Dil-acLDL and BS-1 lectin. EPC Migration Test The migratory function of late EPCs was evaluated by a modified Boyden chamber assay (Transwell, Coster). 10 Briefly, isolated EPCs were detached as described above with trypsin/edta and then late EPCs were placed in the upper chambers of 24-well Transwell plates with polycarbonate membrane (8-µm pores) with serum-free endothelial growth medium; VEGF (50 ng/ml) in medium was placed in the lower chamber. After incubation for 24 h, the membrane was washed briefly with PBS and fixed with 4% paraformaldehyde. The membrane was then stained using hematoxylin solution and carefully removed. The magnitude of migration of the late EPCs was evaluated by counting the migrated cells in six random high-power ( 100) microscopic fields. EPC Tube Formation Assay 8

16 In vitro tube formation assay was performed with an In Vitro Angiogenesis Assay Kit (Chemicon). 10 Briefly, ECMatrix gel solution was thawed at 4 C overnight, mixed with ECMatrix diluent buffer, and placed in a 96-well plate at 37 C for 1 h to allow the matrix solution to solidify. Late EPCs were harvested as described above with trypsin/edta, and then EPCs were placed on a matrix solution with EGM-2 MV medium with MMP-9 inhibitor and incubated at 37 C for 16 h. Tubule formation was inspected under an inverted light microscope ( 100). Four representative fields were taken, and the average of the total area of complete tubes formed by cells was compared by using computer software, Image-Pro Plus. Mouse Aortic Ring Assay for Angiogenesis The mouse aortic ring assay for angiogenesis was performed as previously described. 20 Briefly, the thoracic aorta was isolated and cleaned of perivascular adipose tissue, and 1-mm-long aortic rings were embedded in Matrigel supplemented with 20 U/ml heparin. The aortic rings from wild-type and MMP-9 -/- mice were then cultured in EBM-2 (Cambrex Bioscience) with supplements (hydrocortisone, R 3 -insulin-like growth factor 1, human endothelial growth factor, human fibroblast growth factor, gentamicin, amphotericin B, vitamin C, and 20% fetal bovine serum) and supplemented 9

17 with or without VEGF and recombinant mouse MMP-9 (rmmp-9, Bioclone Inc, San Diego, CA) at 37 C. The number of sprouting microvessels from aortic rings was determined under the microscope once per day for 1 week. Bone Marrow-derived c-kit + cell Isolation Bone marrow-derived MNCs were obtained from wild-type and MMP-9 -/- mice (n=5 for each group). Bone marrow-derived c-kit + cells were isolated by using CD117 MicroBeads and MACS (Miltenyli Biotec GmbH) according to the manufacturer s instruction. C-Kit + BM cells were > 90% positive for CD31 +, and these bone marrow-derived progenitor cells were used for cell functional assays. Bone Marrow Transplantation Model Bone marrow transplantation was performed as previously described. 21 Briefly, recipient MMP-9 -/- mice and wild-type mice at 8 weeks of age were lethally irradiated with a total dose of 9.0 Gy. egfp transgenic mice (FVB background) that ubiquitously expressed enhanced GFP (Level Biotechnology Inc., Taipei, Taiwan) and MMP-9 -/- mice were used as the donors. 21 After being irradiated, 10

18 the recipient mice received unfractionated bone marrow cells ( ) from egfp mice and MMP-9 -/- mice by a tail vein injection. Eight weeks after the bone marrow transplantation, the chimeric mice were rendered a hindlimb ischemic injury (n=8 per group). Repopulation by egfp-positive bone marrow cells was measured by flow cytometry to be 95%. Two weeks after the induction of hindlimb ischemic surgery in the bone marrow-reconstituted mice, tissues were harvested for confocal immunofluorescent and histological analysis. Tissue neovascularization was assessed in frozen sections (5 μm) of the gastrocnemius muscle from the ischemic limbs. Bone marrow-derived EPCs were stained with antibodies directed against egfp (Chemicon) and CD31 (BD PharMingen). EPC density was estimated by counting egfp + CD31 + double-positive cells (yellow color) under HPF ( 100) in at least six different cross-sections from different animals. 11

19 Figure I. Morphology and characterization of human EPCs from peripheral blood. MNCs were plated on a fibronectin-coated culture dish on the first day (A). Four days after plating, adherent early EPCs with a spindle shape were shown (B). Three weeks after plating, late EPCs with a cobblestone-like morphology were selected, reseeded, and grown to confluence (C). Phenotyping of endothelial characteristics of colony-forming units of EPCs (D F). A colony of EPCs was defined as a central core of round cells with elongated sprouting cells at the periphery (D). Most cells were shown to simultaneously endocytose DiI-acLDL (red) (E) and bind fluorescein isothiocyanate UEA-1 (lectin) (green) (F). Immunofluorescence detection (green) of VE-cadherin (G), PECAM-1 (H), and CD34 (I) for late EPCs. Cells were counterstained with propidium iodide for nucleus (red). Scale bar: 50 µm for A-F, 20 µm for G-I. 12

20 Figure II. Schematic representation of ischemia-induced neovascularization, illustrating the role of MMP-9. The process of neovascularization consisted of multiple steps, including EPC mobilization, homing to ischemic tissue, transendothelial migration, and new vessel formation by circulating EPCs and preexisting endothelial cells. Tissue ischemia promotes cytokine stimulation and enhances MMP-9 activation in bone marrow (BM) and ischemic tissue. MMP-9 activation in BM cells can promote BM-derived EPCs to mobilize and infiltrate in ischemic tissues. Also, MMP-9 can function to clear the matrix surrounding endothelial cells in ischemic tissue, and directly contribute to vasculogenesis by promoting the invasive and migratory activities of EPCs. 13