Astrocytic neogenin/netrin-1 pathway promotes blood vessel homeostasis and function in mouse cortex
Ling-Ling Yao, Jin-Xia Hu, Qiang Li, Daehoon Lee, Xiao Ren, Jun-Shi Zhang, Dong Sun, Hong-Sheng Zhang, Yong-Gang Wang, Lin Mei, Wen-Cheng Xiong
Ling-Ling Yao, Jin-Xia Hu, Qiang Li, Daehoon Lee, Xiao Ren, Jun-Shi Zhang, Dong Sun, Hong-Sheng Zhang, Yong-Gang Wang, Lin Mei, Wen-Cheng Xiong
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Research Article Angiogenesis Neuroscience

Astrocytic neogenin/netrin-1 pathway promotes blood vessel homeostasis and function in mouse cortex

Abstract

Astrocytes have multiple functions in the brain, including affecting blood vessel (BV) homeostasis and function. However, the underlying mechanisms remain elusive. Here, we provide evidence that astrocytic neogenin (NEO1), a member of deleted in colorectal cancer (DCC) family netrin receptors, is involved in blood vessel homeostasis and function. Mice with Neo1 depletion in astrocytes exhibited clustered astrocyte distribution and increased BVs in their cortices. These BVs were leaky, with reduced blood flow, disrupted vascular basement membranes (vBMs), decreased pericytes, impaired endothelial cell (EC) barrier, and elevated tip EC proliferation. Increased proliferation was also detected in cultured ECs exposed to the conditioned medium (CM) of NEO1-depleted astrocytes. Further screening for angiogenetic factors in the CM identified netrin-1 (NTN1), whose expression was decreased in NEO1-depleted cortical astrocytes. Adding NTN1 into the CM of NEO1-depleted astrocytes attenuated EC proliferation. Expressing NTN1 in NEO1 mutant cortical astrocytes ameliorated phenotypes in blood-brain barrier (BBB), EC, and astrocyte distribution. NTN1 depletion in astrocytes resulted in BV/BBB deficits in the cortex similar to those in Neo1 mutant mice. In aggregate, these results uncovered an unrecognized pathway, astrocytic NEO1 to NTN1, not only regulating astrocyte distribution, but also promoting cortical BV homeostasis and function.

Authors

Ling-Ling Yao, Jin-Xia Hu, Qiang Li, Daehoon Lee, Xiao Ren, Jun-Shi Zhang, Dong Sun, Hong-Sheng Zhang, Yong-Gang Wang, Lin Mei, Wen-Cheng Xiong

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Figure 8

Increased EC proliferation and migration in HUVEC cultures exposed to Neo1-KO astrocytes or their CM.

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Increased EC proliferation and migration in HUVEC cultures exposed to Ne...
(A–C) Increased EC proliferation in HUVECs cocultured with Neo1-KO astrocytes. (A) Schematic of coculture of HUVECs with cortical astrocytes of control and Neo-KO. BrdU was incubated for 4 hours. (B) Representative images of BrdU+ HUVECs. (C) Quantitative analysis of data in B. Data are represented as mean ± SEM (n = 4 experiments of astrocytes from NeoGFAP-CreER with 4-OH-TAM). (D–F) Increased EC proliferation in HUVECs exposed to CM of Neo1-KO astrocytes. (D) Schematic of the culture. BrdU was incubated for 6 hours. (E) Representative images of BrdU+ HUVECs. (F) Quantification analysis of data in E. Data are represented as mean ± SEM (n = 4 experiments with astrocytes from NeoGFAP-CreER [+TAM] and its control group). (G–K) Increased EC migration in HUVECs exposed to CM of Neo1-KO astrocytes. (G) Schematic of HUVEC migration assay. The HUVECs were cultured with CM of astrocytes in a 6-well plate (left panel), and were scratched for the wound-healing migration assay (right panel). (H) Representative images of HUVEC migration 24 hours after wound scratching. (I) Representative images of HUVECs that were cultured with CM of astrocytes and immunostained with indicated antibodies. (J) Quantification analyses of data in H. Data are represented as mean ± SEM (n = 4 experiments from NeoGFAP-CreER and its control group astrocytes). (K) Quantification of data in I. Data are represented as mean ± SEM (n = 3 experiments from NeoGFAP-CreER and its control group astrocytes). Scale bars: 20 μm. *P < 0.05; **P < 0.01, Mann-Whitney U test.