Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis
Yun He, … , Kari Alitalo, Wang Min
Yun He, … , Kari Alitalo, Wang Min
Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2344-2355. https://doi.org/10.1172/JCI28123.
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Research Article Angiogenesis

Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis

Abstract

Bmx/Etk non-receptor tyrosine protein kinase has been implicated in endothelial cell migration and tube formation in vitro. However, the role of Bmx in vivo is not known. Bmx is highly induced in the vasculature of ischemic hind limbs. We used both mice with a genetic deletion of Bmx (Bmx-KO mice) and transgenic mice expressing a constitutively active form of Bmx under the endothelial Tie-2 enhancer/promoter (Bmx-SK-Tg mice) to study the role of Bmx in ischemia-mediated arteriogenesis/angiogenesis. In response to ischemia, Bmx-KO mice had markedly reduced, whereas Bmx-SK-Tg mice had enhanced, clinical recovery, limb perfusion, and ischemic reserve capacity when compared with nontransgenic control mice. The functional outcomes in these mice were correlated with ischemia-initiated arteriogenesis, capillary formation, and vessel maturation as well as Bmx-dependent expression/activation of TNF receptor 2– and VEGFR2-mediated (TNFR2/VEGFR2-mediated) angiogenic signaling in both hind limb and bone marrow. More importantly, results of bone marrow transplantation studies showed that Bmx in bone marrow–derived cells plays a critical role in the early phase of ischemic tissue remodeling. Our study provides the first demonstration to our knowledge that Bmx in endothelium and bone marrow plays a critical role in arteriogenesis/angiogenesis in vivo and suggests that Bmx may be a novel target for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.

Authors

Yun He, Yan Luo, Shibo Tang, Iiro Rajantie, Petri Salven, Matthias Heil, Rong Zhang, Dianhong Luo, Xianghong Li, Hongbo Chi, Jun Yu, Peter Carmeliet, Wolfgang Schaper, Albert J. Sinusas, William C. Sessa, Kari Alitalo, Wang Min

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Figure 6

Characterization of infiltrated immune cells, gene expression, and Bmx/TNFR2/VEGFR2 signaling in response to ischemia.

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Characterization of infiltrated immune cells, gene expression, and Bmx/T...
(A–D) Recruitment of macrophages/lymphocytes in response to ischemia as determined by anti-F4/80 and anti-CD3 antibodies, respectively. Representative images of nonischemic and ischemic hind limbs in C57BL/6 mice on day 3 are shown for macrophages (A) and for lymphocytes (C). F4/80- and CD3-positive cells were counted as number infiltrating per millimeter squared muscle area (B and D). (E) The expression of proinflammatory and proangiogenic genes was assessed by qRT-PCR in the gastrocnemius 0, 3, 7, 14, and 28 days after ischemia in C57BL/6 mice. (F) Ischemia-induced gene expression in Bmx-KO and Bmx-SK-Tg mice on day 3 (early-responsive genes) or day 28 (late-responsive genes, VEGFR2, Tie-2, and PDGFR-β) after ischemia was determined by qRT-PCR. Data represent the fold increases of each gene, with expression in C57BL/6 mice taken as 1.0. n = 3 for each strain per time point. (G) Bmx is critical for ischemia-induced expression/activation of TNFR2 and VEGFR2. Day 7 postsurgery samples were used for protein analyses. Expression of TNFR2 and phosphorylation of Bmx (pY40), VEGFR2 (pY1154/1159), and p38 were determined by Western blotting with respective antibodies. β-Tubulin protein was used for normalization. (H) Ischemia-induced gene expression in bone marrow of WT, Bmx-KO, and Bmx-SK-Tg mice on day 3 and day 28 after ischemia was determined by qRT-PCR. Data represent the fold increases of each gene, with expression in WT mice without ligation taken as 1.0. n = 3 for each strain per time point.