Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors
Pouneh Kermani, Dahlia Rafii, David K. Jin, Paul Whitlock, Wendy Schaffer, Anne Chiang, Loic Vincent, Matthias Friedrich, Koji Shido, Neil R. Hackett, Ronald G. Crystal, Shahin Rafii, Barbara L. Hempstead
Pouneh Kermani, Dahlia Rafii, David K. Jin, Paul Whitlock, Wendy Schaffer, Anne Chiang, Loic Vincent, Matthias Friedrich, Koji Shido, Neil R. Hackett, Ronald G. Crystal, Shahin Rafii, Barbara L. Hempstead
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Article Angiogenesis

Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors

Abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels.

Authors

Pouneh Kermani, Dahlia Rafii, David K. Jin, Paul Whitlock, Wendy Schaffer, Anne Chiang, Loic Vincent, Matthias Friedrich, Koji Shido, Neil R. Hackett, Ronald G. Crystal, Shahin Rafii, Barbara L. Hempstead

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BDNF accelerates the revascularization of the ischemic limbs. (A and B) ...
BDNF accelerates the revascularization of the ischemic limbs. (A and B) Western blot analysis of tissue lysates at 5 and 7 days after ligation using polyclonal anti-BDNF (Santa Cruz Biotechnology) (A), followed by a chemiluminescence-based detection, ECL and immunohistochemical detection of tissue sections to assess BDNF expression (B) in ischemic (Isch.) or non-ischemic (Cont.) limb at day 7 after ligation. BDNF expression was detected using frozen sections, anti-BDNF, and a VIP substrate, resulting in a red-purple reaction product. Magnification, ×10. (C) Immunohistochemical detection of phosphorylated TrkB in mouse muscle sections, in ischemic (Isch.) or non-ischemic (Cont.) limb, 21 days after vessel ligation. The boxes in the lower right corners of B and C represent the negative controls. (D) Hindlimb blood flow monitored serially for 3–21 days after ligation in mice receiving 1 × 108 PFU AdBDNF (red), AdVEGF-A (green), AdNull (black), or PBS (blue). Blood flow is calculated as the ratio of flow in the ischemic limb to that in the non-ischemic limb. Values are expressed as the mean ± SEM for 5 animals per condition. (E) Immunohistochemical analysis, 21 days after ligation, of quadriceps muscle sections from animals receiving the indicated treatment (above columns). Endothelial cells, smooth muscle cells, and monocytes/macrophages were identified using antibodies against CD31, smooth muscle cell α-actin (arrows), and MOMA-2 (arrows), respectively. Magnification, ×40. Sections were analyzed and photographed by investigators “blinded” to sample identity and are representative of tissue obtained from 5 animals evaluated per group.