Abstract
Background and Purpose—Bone marrow–derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)–induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng+/−) mice. We hypothesized that Eng+/− BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation.
Methods—BM transplantation was performed using adult wild-type (WT) and Eng+/− mice as donors/recipients. An adeno-associated viral vector expressing VEGF was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 µm/100 vessels), and BMDCs in the angiogenic foci were analyzed.
Results—The dysplasia index of WT/Eng+/− BM mice was higher than WT/WT BM mice (P<0.001) and was similar to Eng+/−/Eng+/− BM mice (P=0.2). Dysplasia in Eng+/− mice was partially rescued by WT BM (P<0.001). WT/WT BM and WT/Eng+/− BM mice had similar numbers of BMDCs in the angiogenic foci (P=0.4), most of which were CD68+. Eng+/− monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1.
Conclusions—Endoglin-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations.
Introduction
Mutations in human endoglin (ENG) cause hereditary hemorrhagic telangiectasia 1. Telangiectases and arteriovenous malformations have been viewed as a disorder of the extant endothelium.1 Vascular endothelial growth factor (VEGF) induced cerebrovascular dysplasia in adult Eng heterozygous (Eng+/−) mice2; the majority of bone marrow–derived cells (BMDCs) in the angiogenic foci were monocytes/macrophages (Mø),3 which contribute to vascular repair and angiogenesis.4 We hypothesized that Eng deficiency in BMDCs causes cerebrovascular abnormalities in mice after VEGF stimulation.
Methods
After institutional approval, the design and groups are listed in Figure I and Figure II in the online-only Data Supplement and methods described in the online-only Data Supplement were used.
Results
Adeno-associated viral vector expressing VEGF induced brain angiogenesis in all groups and caused abnormal cerebrovascular morphology in mice with Eng+/− BM (Figure 1A). Vascular densities (mean±SD) were as follows: 820±153 (wild type [WT]/WT BM), 720±150 (Eng+/−/WT BM), 653±120 (WT/Eng+/− BM), and 674±76 vessels/mm2 (Eng+/−/Eng+/− BM). Mice carrying Eng+/− somatic or BM cells showed a trend toward lower vascular density compared with WT/WT BM mice (P=0.06; Figure 1B). WT/Eng+/− BM mice had >5-fold greater dysplasia index than WT/WT BM mice (1.7±0.3 versus 0.3±0.3; P<0.001; Figure 1C), comparable with the dysplasia index of Eng+/−/Eng+/− BM mice (1.9±0.4; P=0.2). Transplantation of WT BM to Eng+/− mice partially rescued dysplasia (P<0.001; Figure 1C).
VEGF induced cerebrovascular dysplasia in mice with Eng+/− BM. A, Representative images of VEGF-induced brain angiogenic foci. Arrows indicate dysplastic vessels. Scale bar, 50 µm. Quantifications of (B) vascular density and (C) dysplasia index. *P<0.001 compared with WT/WT BM group; #P<0.001 compared with Eng+/−/WT BM group. Data are represenred as mean±SD (n=6 per group). BM indicates bone marrow; Eng+/, endoglin heterozygous; VEGF, Vascular endothelial growth factor; and WT, wild type.
Using enhanced green fluorescent protein–expressing donors, we found that WT/WT BM and WT/Eng+/− BM mice had similar BMDC counts in the angiogenic foci (400±125 versus 339±112/mm2; P=0.4; Figure SIIIA and SIIIB in the online-only Data Supplement). The majority of BMDCs was CD68+ (WT/WT BM: 67%±8 versus WT/Eng+/− BM: 64%±10; P=0.6; Figure 2A and 2C; Figure SIIIC in the online-only Data Supplement). Approximately 7% of BMDCs in both groups were CD31+ endothelial cells (ECs Figure 2B and 2D).
Eng deficiency did not alter BMDC homing ability. A and C, Most of the recruited GFP+ BMDCs were CD68+ Mø (arrows). B and D, Few GFP+ BMDCs were CD31+ ECs (arrows). Scale bars, 50 µm in (A) and 20 µm in (B). Data are represented as mean±SD (n=6 per group). BMDC indicates bone marrow-derived cell; Eng, endoglin; and GFP+, green fluorescent protein.
BM-derived Mø from WT and Eng+/− mice were cultured and treated with 4 doses of VEGF (0, 10, 50, and 100 ng/mL) for 18 hours. Compared with WT, Eng expression was 50% lower in Eng+/− Mø (Figure SIVA in the online-only Data Supplement). The presence of both Vegfr1/Flt1 and Vegfr2/Flk1/Kdr indicates that Mø can be stimulated by VEGF (Figure SIVB and SIVC in the online-only Data Supplement). Matrix metalloproteinase-9 was upregulated in WT but not Eng+/− cells at 50 ng/mL of VEGF (P=0.003; Figure 3A). Notch1 expression in Eng+/− Mø decreased at 100 ng/mL of VEGF treatment compared with WT (P<0.001; Figure 3B).
Mmp9 and Notch1 expression were reduced in Eng+/− monocytes/Mø after VEGF stimulation. Quantification of (A) Mmp9 and (B) Notch1 expression. Expression levels are relative to that of WT–untreated cells. Data are represented as mean±SD from 3 independent experiments (n=3 per group). *P<0.05. Metalloproteinase-9 (Mmp9) and Notch1 expression were reduced in endoglin heterozygous (Eng+/−) vascular endothelial growth factor (VEGF) wild-type (WT)–
Discussion
This is the first demonstration that Eng haploinsufficiency in BMDCs was sufficient to cause cerebrovascular dysplasia in the adult mouse after angiogenic stimulation. The abnormal angiogenic response was associated with altered expression of angiogenesis-related genes in Mø. These findings are consistent with previous work in Eng+/− myocardial infarction mice showing that transfusion of normal, but not hereditary hemorrhagic telangiectasia 1, human mononuclear cells rescued the defect.4
Transforming growth factor-β, VEGF, and Notch pathways act either synergistically with or antagonistically against each other during angiogenesis in a context-dependent manner.5 Notch signaling in Mø plays a critical role in angiogenesis and repair. Abrogation of monocytic Notch1 adversely affected repair after myocardial injury.6 Conditional deletion of Mø Notch1 caused abnormal anastomosis between angiogenic sprouts.7 Further study is needed to examine whether reduced Notch1 signaling in Eng+/− Mø contributes to a dysplastic phenotype.
VEGF dose-dependent effect on Mø depends on culture conditions. Chemotactic response of human Mø to VEGF peaked at 12 ng/mL and decreased after 40 ng/mL with 2-hour incubation.8 We found that 50 ng/mL of VEGF upregulated metalloproteinase-9 in murine Mø, whereas neither 10 nor 100 ng/mL had any effect. Possible explanations are as follows: (1) human cells respond to VEGF differently from mouse cells; and (2) various VEGF doses differentially trigger various signaling pathways to regulate diverse monocytic functions. Notch1 is induced by VEGF in arterial ECs.5 However, its expression in mouse Mø was not affected by VEGF (10–100 ng/mL) in our study, possibly because only a subpopulation of Mø expresses Notch1 during angiogenesis.7
Growth factors and cytokines produced by BMDCs can affect local angiogenesis via systemic signaling. We showed that the mobilization of metalloproteinase-9–deficient BMDCs into circulation in response to VEGF was reduced, which resulted in less BMDC homing and brain angiogenesis.9 VEGF may affect Mø polarization by effects on Notch signaling.10 Further studies should address the indirect/systemic effects of Eng deficiency on the BMDC function and the effect of VEGF on Mø polarization.
Eng deficiency in endothelial precursors may also play a role and deserves further study. In tumors, very few endothelial precursors are capable of triggering the angiogenic switch.11 Furthermore, only a small number of homozygously Eng-deleted ECs (≈1%) was sufficient to induce macroscopic cerebrovascular dysplasia after VEGF stimulation.12
In summary, ≥1 subpopulations of Eng+/− BMDCs are sufficient to induce an abnormal vascular response to brain angiogenic stimulation. Highly relevant to hereditary hemorrhagic telangiectasia 1, it may be possible to envision development of a rescue strategy using BM transplantation therapy. The role of BMDCs in sporadic brain arteriovenous malformations needs further study, because Mø are associated with lesion13 and endothelial precursors incorporate into the abnormal vascular structures.14 Consideration should also be given to the role of ENG in other cerebrovascular diseases, such as stroke.
Acknowledgments
We thank Jeffrey Nelson for statistical consultation, Voltaire Gungab for manuscript preparation, and University of California, San Francisco brain arteriovenous malformation project members (http://avm.ucsf.edu/faculty_staff/) for support.
Sources of Funding
This work was supported by grants from National Institutes of Health (R01NS027713 to W.L. Young, R21NS070153 to H. Su, and P01NS044155 to W.L. Young and H. Su), American Heart Association (SDG0535018N to H. Su), Leslie Munzer Foundation (to H. Su), The Aneurysm and AVM Foundation (TAAF) (to H. Su), and Michael Ryan Zodda Foundation (to W.L. Young and J. Pile-Spellman).
Disclosures
None.
Footnotes
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.112.671974/-/DC1.
- Received July 23, 2012.
- Revision received October 31, 2010.
- Accepted November 19, 2012.
- © 2013 American Heart Association, Inc.
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- Endoglin Deficiency in Bone Marrow is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse After Vascular Endothelial Growth Factor Stimulation
