Trkb signaling in pericytes is required for cardiac microvessel stabilization

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development

BDNF regulates the morphology and migration of human umbilical smooth muscle cells (HuSMCs).

<p>(A) Immunostaining of HuSMCs with TrkB antibody. Scale bar = 10 µm. (B) Western blot analysis for TrkB expression in HuSMCs. Lysate of rat cortical neurons in culture for 6 days was used as positive control for TrkB expression. (C) Phase contrast micrographs at different time points of HuSMCs treated with BDNF, and K252a+BDNF. and BDNF, The arrow heads represent membrane protrusions induced by BDNF. Scale bar = 20 µm. (D) Quantification of (C), and reactivity of HuSMCs after treatment with Y27632 (ROCK inhibitor) and BDNF, or C3 transferase (Rho inhibitor) and BDNF. n control = 9; n BDNF = 8; n K252a+BDNF = 7; n Y27632+BDNF = 10; n C3+BDNF = 7. (E) Scratch assay showing BDNF-induced HuSMCs migration 13 h after the addition of the ligand. The experiment was repeated in triplicate in 3 independent experiments. Bars represent mean ± s.e.m. Statistical comparisons were made by one-way analysis of variance test with Duncan’s <i>post hoc</i> analysis. *P<0.05. (F) Western blot analysis showing reduced expression of TrkB after shRNA knockdown.</p

Microvessel abnormalities in <i>trkb^−/−</i> embryos.

<p>(A–D) Immunofluorescent staining using IB-4 (green) to detect endothelial cells and PDGFRβ (red) to detect pericytes, within the ventricular wall in wild type (A–C) and <i>trkb^−/−</i> (B–C) littermates at E17.5. High magnification images at 100X for wild type (C) and <i>trkb^−/−</i> (D). The PDGFRβ staining is discontinuous and lacks close apposition to IB-4 in the <i>trkb^−/−</i> embryos (arrow) as compared to the wild type embryos. Scale bar A, B = 20 µm. Scale bar C, D = 10 µm. (E) Quantification of (A–D). Bars represent mean ± s.e.m. n = 3 per group. Statistical comparisons were made by one-way analysis of variance test. *P<0.05. PDGFRβ immunoreactivity in the <i>trkb^−/−</i> hearts is reduced, as compared to the wild type hearts. (F,G) Electron-micrographs of ventricles from wild type (F) or <i>trkb^−/−</i> (G) embryos at E17.5. Endothelial cells frequently exhibited degenerated plasma membrane (arrow head) and perivascular edema was consistently detected in capillaries and arterioles of <i>trkb^−/−</i> embryos. Scale bar F,G = 2 µm.</p

Ventricular wall thinning and hemorrhage in <i>trkb^−/−</i> embryos.

<p>Embryos at the indicated gestational age were harvested from <i>trkb^+/−</i> females. (A) Sections of the heart were analyzed following hematoxylin and eosin staining. Ventricular wall size was normal in mid-gestation in the wild type animals, but thinning became apparent by E16.5 in the <i>trkb^−/−</i> embryos. The square inset represent the region were the ventricular wall was quantified in (B). Scale bar = 300 µm. (B) Quantification of (A). Bars represent mean ± s.e.m. n = 3 per group, where we analyzed 6 sections per animal. Statistical comparisons were made by one-way analysis of variance test. *P<0.05. (C) Sections of the right ventricular wall were analyzed histologically following hematoxylin and eosin staining. Intramyocardial hemorrhage from <i>trkb^−/−</i> embryos could be detected at E16.5 (arrowheads) and was absent in wild type littermates. Scale bar = 50 µm.</p

Pericyte/SMCs-specific deletion of TrkB induces a reduction in pericyte density.

<p>(A) Double immunofluorescence staining of the left ventricular wall from P21 mice. TrkB (red) is expressed by perivascular cells (PECAM in green) in wild type mice and is largely reduced in <i>trkb^f/f-</i>SMCCre+ littermates. (B–G) Quantification of NG2+ (B, C, D) and PECAM+ (E, F, G) cells and intensity in the heart. <i>n = 3 per genotype.</i> Bars represent mean ± s.e.m. Statistical comparisons were made by one-way analysis of variance test. <i>(C) *: P = 0.0023. (D) *: P = 0.007. (F) *: P = 0.0022.</i> Scale bar A,B and E = 20 µm.</p

Signaling pathways involved in TrkB-BDNF-induced migration of HuSMCs.

<p>(A) ERK1/2 phosphorylation 30 minutes after BDNF administration to HuSMCs. (B) Quantification of (A) normalized to total ERK. Bars represent mean ± s.e.m. n = 3 per group. Statistical comparisons were made by one-way analysis of variance test. *P<0.05. (C) phosphor-MLC (green) after 1 hour of BDNF addition to HuSMCs. Representative images of 3 independent experiments. Scale bar = 20 µm. (D) Western blot showing MLC phosphorylation after 1 hour of BDNF addition (with or without the Y27632 ROCK inhibitor) to HuSMCs. (E) Quantification of (D) normalized to total MLC. Bars represent mean ± s.e.m. n = 3 per group. Statistical comparisons were made by one-way analysis of variance test with Duncan’s <i>post hoc</i> analysis. *P<0.05. (F–G) Proposed model of TrkB/BDNF signaling role in the cardiac microvasculature. (F) When TrkB/BDNF signaling is present, pericytes migrate in an ERK1/2, Rho/ROCK, and MLC-dependent manner to ensheath the endothelial cells in capillaries. This pericyte coverage of the endothelium promotes mature and stable capillaries. (G) In the absence of TrkB/BDNF signaling, pericytes do not cover the nascent vessels and the resulting capillaries are unstable and leaky.</p