Bone Marrow-Derived Cells Contribute to Vascular Endothelial Growth Factor–Induced Angiogenesis in the Adult Mouse Brain by Supplying Matrix Metalloproteinase-9
Background and Purpose—Our previous studies have shown that bone marrow-derived cells (BMDCs) home to a brain angiogenic focus. The angiogenic response to vascular endothelial growth factor (VEGF) stimulation is reduced in matrix metalloproteinase-9 (MMP-9) knockout mice. We hypothesized that BMDCs contribute to VEGF-induced angiogenesis by supplying MMP-9.
Methods—Bone marrow (BM) transplantation was conducted using MMP-9 knockout (MMP-9 KO) or wild-type (WT) mice as both donors and recipients. Adeno-associated viral vectors expressing VEGF or LacZ were injected into the striatum 4 weeks after BM transplantation. Circulating white blood cells (WBCs), microvessel density, number of BMDCs, and MMP-9 activity around the injection site were analyzed.
Results—Two weeks after vector injection, circulating WBCs increased in WT mice but not in MMP-9 KO mice. VEGF overexpression increased microvessel density by 38% in WT mice 4 weeks after vector injection (P=0.0001). After transplantation of MMP-9 KO BM to WT mice, microvessel density only increased 18% after VEGF stimulation (P=0.037), with MMP-9 activity reduced to 35% of the level of WT mice (P<0.01). There was minimal angiogenic response in MMP-9 KO mice with MMP-9 KO BM (P=0.28). Transplantation of WT BM to MMP-9 KO mice restored brain angiogenic response to 92% of the WT level, ie, a 30% increase of microvessel density with VEGF overexpression (P=0.0006); MMP-9 activity was similar to that in WT mice.
Conclusions—BM-derived MMP-9 plays an important role in BM cell mobilization and focal angiogenesis in the brain in response to VEGF stimulation.
- bone marrow-derived cells
- brain angiogenesis
- vascular endothelial growth factor
Increasing evidence indicates that bone marrow-derived cells (BMDC) are associated with angiogenesis-related disorders, including tumor1,–,3 and brain arteriovenous malformations.4 BMDC are recognized as a major supplier of matrix metalloproteinases (MMP),2,5 a family of zinc-dependent endopeptidases responsible for the degradation of extracellular matrix component, for tissue angiogenesis.
Our previous studies have demonstrated that cerebral angiogenesis can be induced by adeno-associated viral vector (AAV)-mediated vascular endothelial growth factor (VEGF) gene transfer.6,7 This angiogenic response to VEGF stimulation was reduced in MMP-9 knockout (KO) mice,7 suggesting that MMP-9 plays a role in cerebral angiogenesis.
Studies have shown that MMP-9 facilitates mobilization of bone marrow (BM) cells into the circulation8 and their homing to the tumorangiogenic foci.3 In this study, we provide direct evidence that BMDC-derived MMP-9 is crucial for VEGF-induced brain angiogenesis.
Our primary motivation for undertaking this study of the association of BM-derived MMP-9 and angiogenesis is in the context of various brain vascular malformations, especially brain arteriovenous malformations. The other neurological diseases, including tumor, are secondary areas of potential relevance.
Materials and Methods
The experimental design and groups are shown in Figure 1 and the Table. All experimental procedures for using laboratory animals were approved by the Institutional Animal Care and Use Committee, University of California, San Francisco. Adult male mice at age 8 to 10 weeks in the C57BL/6 background carrying homozygous null mutations of the MMP-9 gene9 (a gift from Dr. Zena Werb, Depertment of Anatomy, University of California, San Francisco, CA) and their wild-type (WT) littermates were used as BM donors and recipients in all the experiments. For Methods and Statistical Analysis, please see Supplemental Methods (available online at http://stroke.ahajournals.org).
MMP-9 Facilitates the Mobilization of BM Cells Into the Circulation
WT+WT BM and MMP-9 KO+KO BM mice were used to explore the effects of MMP-9 on BM cell mobilization in response to VEGF stimulation. The mice in these 2 groups had a similar baseline of total circulating white blood cells (WBC). Total WBC increased to 160% of baseline (P=0.0015) in WT+WT BM mice 14 days after intrabrain injection of AAV-VEGF, decreased thereafter, and then returned to baseline on day 25 (Figure 2A). The total WBC increased slightly on day 8 only (135% of baseline; P=0.08) in MMP-9 KO+KO BM mice. Thus, VEGF could not induce a significant increase in total WBC in KO+KO BM mice.
Both neutrophils and lymphocytes peaked on day 14 and returned to baseline on day 25 in VEGF-treated WT+WT BM mice (Figure 2B, C). They were the major cell types responsible for the increase of total WBC at this time point. These cells did not increase in KO+KO BM mice. Monocytes increased in WT+WT BM mice but not in KO+KO BM mice on day 14 after AAV-VEGF and AAV-LacZ vector injection (Figure 2D). These data suggest that MMP-9 facilitates BM cell mobilization in response to VEGF stimulation.
VEGF Treatment Induces MMP-9 Expression in BM Cells
We then determined whether the increased mobilization of BM cells in VEGF-treated WT+WT BM mice was attributable to the upregulation of MMP-9 in the BM cells. We analyzed MMP-9 expression in BM tissue collected from WT+WT BM and MMP-9 KO+KO BM mice. Very few MMP-9-positive cells were detected in the BM of LacZ-treated WT+WT BM mice (data not shown). MMP-9-positive signals were detected in BM sections of VEGF-treated WT+WT BM mice (Figure 2E, parts c and d), but not in KO+KO BM mice (Figure 2E, parts a and b). These results suggest that VEGF upregulated MMP-9 expression in BM cells.
MMP-9 Promotes the Recruitment of BMDC to the Angiogenic Foci
We then explored the role of MMP-9 in the recruitment of BMDC from the circulation to the angiogenic foci in the mouse brain. The brain sections were stained with an antibody specific to CD45, a common leukocyte marker. CD45+ cells were observed in the VEGF-treated brain (Figure 3B, D), but not in the LacZ-treated brain (Figure 3A, C). Numerous CD45+ BMDC were observed in the WT+WT BM brain around the injection site (Figure 3D, E, and F; 101±37/hemisphere section), but few were detected around the injection site in the brain of MMP-9 KO+KO BM mice (Figure 3B, F; 26±9/hemisphere section), indicating that MMP-9 promoted the recruitment of BMDC (CD45+ cells) into the VEGF-induced angiogenic foci.
Transplantation of WT BM Rescued the Impaired Brain Angiogenesis of MMP-9 KO Mice
Microvessel density increased 38% in the brain of VEGF-treated WT+WT BM mice (Figure 4C, D; 318±44 vs 230±16, VEGF vs LacZ; P=0.0001) and 18% in VEGF-treated WT+KO BM mice (Figure 4C; 248±13 vs 210±34, VEGF vs LacZ; P=0.037). VEGF did not induce significant angiogenesis in the brain of KO+KO BM mice (231±31 vs 211±40, VEGF vs LacZ; P=0.28). Transplantation of WT BM partially rescued the impaired angiogenic response in MMP-9 KO mice, indicated by an increase of microvessel density to 30% in the brain of AAV-VEGF-injected mice compared to that of AAV-LacZ-injected mice (Figure 4C; 278±19 vs 213±29, VEGF vs LacZ; P=0.0006). These results suggest that BMDC homing is crucial for VEGF-induced brain focal angiogenesis.
We then used antibodies specific to CD31 and cell proliferating marker BrdU to show the proliferating endothelial cells. The numbers of BrdU/CD31-double-positive cells in each group paralleled the data for microvessel density. There were more double-positive cells in KO+WT BM compared to KO+KO BM mice and fewer double-positive cells in WT+KO BM compared to WT+WT BM mice (Figure 4E).
We also analyzed the correlation of microvessel density with injected vectors, recipient genotypes, and donor BM genotypes using 3-way ANOVA. We observed that injection of AAV-VEGF increased microvessel density (P=0.0001; AAV-VEGF vs AAV-LacZ). VEGF was more effective in WT+WT BM mice compared to MMP-9 KO +KO BM mice (P=0.0642) and WT mice with MMP-9 KO BM (P=0.0037) genotypes. Linear regression revealed that both recipient (P=0.022) and donor BM (P=0.002) genotypes influenced the microvessel density in the VEGF-treated brain (Figure 4C).
BMDC Are Important Sources of MMP-9 in the Brain Angiogenic Foci
To investigate whether BMDC are important sources of MMP-9 in VEGF-induced angiogenic foci in the mouse brain, we examined the level of MMP-9 activity in the VEGF-treated brain using zymography. A higher level of MMP-9 was detected in VEGF-treated WT+WT BM mice (3.7±2.2) than in MMP-9 KO+KO BM mice (1.0±0.1; Figure 5; P<0.01). The MMP-9 level was significantly reduced in WT+KO BM mice (1.3±0.3) but was greatly increased in KO mice that underwent transplantation with WT BM (3.8±1.1; Figure 5; P<0.01). These results suggest that BMDC provided a significant amount of MMP-9 to the VEGF-induced brain angiogenic foci.
This study is the first to our knowledge to show that after VEGF administration in the adult mouse brain: (1) circulating WBC increased in WT+WT BM mice (accompanied by MMP-9 expression in the BM) but not in MMP-9 KO+KO BM mice, suggesting that MMP-9 activation facilitates mobilization; (2) numerous BMDC (CD45+ cells) were observed in the brain of WT+WT BM mice and few were observed in the brain of KO+KO BM mice, suggesting that MMP-9 promotes the recruitment of BMDC to VEGF-induced angiogenic foci; and (3) focal brain angiogenesis decreased in WT mice that underwent transplantation with KO BM, accompanied by a reduced level of brain MMP-9, whereas transplantation of WT BM partially rescued the impaired angiogenic response of MMP-9 KO mice, with an increased level of brain MMP-9. Taken together, our results demonstrate that BM-derived MMP-9 facilitates the mobilization of BM cells into the circulation and their homing to the angiogenic foci in response to VEGF stimulation. BMDC contribute to angiogenesis by supplying MMP-9.
MMP-9 Is Crucial for VEGF-Induced BM Cell Mobilization
Stem cells are quiescent in the BM niche, but they are mobilized to the peripheral blood in response to specific signals. Single-dose intravenous injection of Adenoviral vector (Ad)-VEGF increases circulating WBC in WT mice, which is MMP-9-dependent and is associated with an elevation of VEGF plasma level.8 Our results showed that intrabrain injection of AAV-VEGF increased circulating WBC to 160% of baseline in WT+WT BM mice. This effect was not observed in MMP-9 KO+KO BM mice (Figure 2A). The number of circulating WBC peaked on day 14, likely the peak of plasma VEGF; Heissig et al8 showed in their article that the peak of the mature WBC in circulation correlated with the peak of plasma VEGF level. Further studies are needed to prove this in our model.
Activated MMP-9 within the BM liberates soluble kit-ligand, thereby mobilizing BM cells into the circulation.8,10 We showed that injection of AAV-VEGF into the mouse brain increased MMP-9 expression in BM cells (Figure 2E), resulting in increased mobilization of BM cells, as demonstrated by increased circulating WBC. Although soluble kit-ligand released by induced MMP-9 in BM cells seems to be the key factor in the process, further studies to investigate whether exogenous soluble kit-ligand rescues impaired BM mobilization in MMP-9 KO mice may provide more evidence.
BMDC Contribute to VEGF-Induced Focal Brain Angiogenesis by Supplying MMP-9
We have previously studied whether BMDC (tracked by green fluorescent protein expression after transplantation of BM cells harvested from green fluorescent protein mice to WT mice) can be recruited into AAV-VEGF-induced angiogenic foci, and whether they incorporate into the neovasculature.7 We demonstrated that MMP-9 activity is necessary for the VEGF-induced focal angiogenesis by injecting AAV-VEGF into the brain of MMP-9 KO mice.7 However, we have no direct evidence to show that BMDC contribute to MMP-9 in the VEGF-induced angiogenic foci.
In the current study, we used a BM transplantation mouse model to test our hypothesis that MMP-9 derived from BMDC is crucial for VEGF-induced brain angiogenesis. We found that transplanting MMP-9 KO BM to WT mice resulted in a reduction of their brain angiogenic response to VEGF stimulation, whereas transplanting WT BM to MMP-9 KO mice partially rescued their brain angiogenic response. Thus, this study provides solid evidence that BMDC-derived MMP-9 contributes to VEGF-induced brain focal angiogenesis. In addition, we demonstrate that MMP-9 facilitates the mobilization of BM cells into the circulation, which subsequently may facilitate their recruitment into the mouse brain angiogenic foci.
To what extent BMDC incorporate into the neovasculature remains controversial. Despite the fact that only small fractions of circulation cells are monocytes, we found that most of BMDC homing to VEGF-stimulated brain angiogenic focus are CD45+ (94%) and CD68+ (71%) cells.7 Others also have found that BMDC infiltrating into the brain give rise primarily to microglia.11,–,14 Studies including ours have shown that BMDC are recruited to the angiogenic foci but are rarely incorporated into the vessel structure,1,7, 15,16 suggesting a paracrine angiogenic role for BMDC.
In this study, transplantation of BM cells from MMP-9 KO mice into WT recipient mice, and vice versa, was conducted to study the effects of BM-derived MMP-9 on VEGF-induced focal brain angiogenesis. Brain angiogenesis indicated by microvessel density decreased in VEGF-treated WT+KO BM mice compared to WT+WT BM mice, accompanied by a decrease in MMP-9 level to 35% of that in WT+WT BM mice; the impaired angiogenesis in MMP-9 KO mice was partially rescued by transplantation of WT BM. A near-normal level of MMP-9 activity was also detected in the brain of MMP-9 KO mice that underwent transplantation with WT BM. These results suggest that BM-derived MMP-9 is crucial for cerebral angiogenesis, and that BMDC are an important source of MMP-9 in the VEGF-induced angiogenic foci. MMP-9 provided by BMDC promotes local angiogenesis by degrading the extracellular matrix, resulting in capillary branching. It also cleaves membrane-bound cytokines17 such as VEGF, which, in turn, promotes angiogenesis.18 Moreover, BMDC themselves express VEGF and other angiogenic factors.19 MMP-9, the major player, delivered by recruited BMDC along with other BMDC-derived proangiogenic factors modulates proliferation of local endothelial cells.
Interestingly, microvessel density slightly increased in the brain of VEGF-treated WT mice that underwent transplantation with MMP-9 KO BM mice, and transplantation of WT BM partially rescued the impaired angiogenesis in MMP-9 KO mice. Our results suggest that although BMDC are the major participant in VEGF-induced focal brain angiogenesis, resident non-BM cells of the recipient brain may compensate partly for the lack of BMDC-derived MMP-9. The statistical analysis confirms that both the somatic genotype and BM genotype influenced the microvessel density in the VEGF-treated brain.
A limitation of our study is that we did not clarify the contribution of various resident cell types in the target brain tissue. We did observe a weak band of MMP-9 in the brain of WT mice that underwent transplantation with MMP-9 KO BM (Figure 5A), which is probably derived from other resident cells in the brain such as neurons, astrocytes, or endothelia. Another limitation of our study is that we did not clarify how BM and the brain communicate, although this phenomenon has been observed by other investigators.20 The possibility exists that the signal from the brain to the BM derives from circulating VEGF. Heissig et al8 have shown that plasma VEGF level increased after Ad-VEGF injection (intravenous). We believe that an increase of plasma VEGF was present in our mice, which will be addressed in our future studies.
The important point that our study makes is that a biologically significant fraction of MMP-9 activity arrives at an angiogenic focus from the extracranial BM site. Not only is this important for understanding the precise mechanisms and procedural considerations for a focal angiogenic response but also it opens up a potential line of inquiry to target this blood-borne source of protease activity for eventual new therapeutic approaches for angiogenic-related neurological disorders.
In this study, we showed that BM-derived MMP-9 plays a key role in VEGF-induced brain angiogenesis, including BM cell mobilization and BMDC recruitment to the angiogenic foci. BMDC contribute to focal angiogenesis by supplying MMP-9. Our study suggests that modifying MMP-9 activity of BMDC may be a therapeutic strategy for the treatment of angiogenic-related disorders, such as brain arteriovenous malformations and brain tumor.
Sources of Funding
This study was supported in part by grants from the National Institutes of Health: R01 NS27713 (W.L.Y.) and P01 NS44155 (W.L.Y., H.S.).
The authors thank Charles E. McCulloch, PhD, for assistance with statistical analysis, Voltaire Gungab for assistance with manuscript preparation, and the other members of the UCSF BAVM Study Project (http://avm.ucsf.edu) for their support.
The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.596452/DC1.
- Received July 12, 2010.
- Accepted August 23, 2010.
- © 2011 American Heart Association, Inc.
- Jodele S,
- Chantrain CF,
- Blavier L,
- Lutzko C,
- Crooks GM,
- Shimada H,
- Coussens LM,
- Declerck YA
- Hao Q,
- Liu J,
- Pappu R,
- Su H,
- Rola R,
- Gabriel RA,
- Lee CZ,
- Young WL,
- Yang GY
- Priller J,
- Flügel A,
- Wehner T,
- Boentert M,
- Haas CA,
- Prinz M,
- Fernández-Klett F,
- Prass K,
- Bechmann I,
- de Boer BA,
- Frotscher M,
- Kreutzberg GW,
- Persons DA,
- Dirnagl U
- Zentilin L,
- Tafuro S,
- Zacchigna S,
- Arsic N,
- Pattarini L,
- Sinigaglia M,
- Giacca M
- Purhonen S,
- Palm J,
- Rossi D,
- Kaskenpää N,
- Rajantie I,
- Ylä-Herttuala S,
- Alitalo K,
- Weissman IL,
- Salven P
- Vu TH,
- Werb Z
- Zacharek A,
- Shehadah A,
- Chen J,
- Cui X,
- Roberts C,
- Lu M,
- Chopp M