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, 48 (9), 2557-2564

Brain Transforming Growth Factor-β Resists Hypertension Via Regulating Microglial Activation


Brain Transforming Growth Factor-β Resists Hypertension Via Regulating Microglial Activation

You Li et al. Stroke.


Background and purpose: Hypertension is the major risk factor for stroke. Recent work unveiled that hypertension is associated with chronic neuroinflammation; microglia are the major players in neuroinflammation, and the activated microglia elevate sympathetic nerve activity and blood pressure. This study is to understand how brain homeostasis is kept from hypertensive disturbance and microglial activation at the onset of hypertension.

Methods: Hypertension was induced by subcutaneous delivery of angiotensin II, and blood pressure was monitored in conscious animals. Microglial activity was analyzed by flow cytometry and immunohistochemistry. Antibody, pharmacological chemical, and recombinant cytokine were administered to the brain through intracerebroventricular infusion. Microglial depletion was performed by intracerebroventricular delivering diphtheria toxin to CD11b-diphtheria toxin receptor mice. Gene expression profile in sympathetic controlling nucleus was analyzed by customized qRT-PCR array.

Results: Transforming growth factor-β (TGF-β) is constitutively expressed in the brains of normotensive mice. Removal of TGF-β or blocking its signaling before hypertension induction accelerated hypertension progression, whereas supplementation of TGF-β1 substantially suppressed neuroinflammation, kidney norepinephrine level, and blood pressure. By means of microglial depletion and adoptive transfer, we showed that the effects of TGF-β on hypertension are mediated through microglia. In contrast to the activated microglia in established hypertension, the resting microglia are immunosuppressive and important in maintaining neural homeostasis at the onset of hypertension. Further, we profiled the signature molecules of neuroinflammation and neuroplasticity associated with hypertension and TGF-β by qRT-PCR array.

Conclusions: Our results identify that TGF-β-modulated microglia are critical to keeping brain homeostasis responding to hypertensive disturbance.

Keywords: blood pressure; diphtheria toxin; hypertension; microglia; stroke.


Figure 1
Figure 1. Brain TGFβ signaling is important in regulating BP in hypertension
Mice were treated with ICV infusion of TGFβ neutralizing antibody (A), TGFβRI inhibitor SB525334 (B) or TGFβ1 (C); hypertension was induced in each group by Ang II infusion. Systolic BP was measured by tailcuff in (A) and (B) and by telemetry transducer in (C). * P<0.05, ** P<0.01.
Figure 2
Figure 2. Effects of brain TGFβ1 treatment on neural activity and humoral expression
(A) Summary semi-quantification of Western blots of NMDAR2A and GAD65 in hypothalamus and brain stem tissues derived from naïve, Ang II- and TGFβ1+Ang II-treated mice. (B) Norepinephrine level was examined by ELISA in kidney lysates from naïve, Ang II- and TGFβ1+Ang II-treated mice. ** P<0.01; *** P<0.001.
Figure 3
Figure 3. TGFβ1 suppresses microglial activation
(A) Representative immunostaining of microglia with anti-Iba1 antibody in the PVN. Calibration bar equals to 20 μm. Fractional area of Iba-1 staining was analyzed in each group. **** P<0.0001. Expression of MHC class II subtype I-Ab (B) and TNFα (C) in CD11b+CD45low-microglia was evaluated by flow cytometry analysis. *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4. TGFβ1 promotes SMAD2/3 phosphorylation in microglia
(A) Flow cytometry analysis of SMAD2/3 phosphorylation in naïve and TGFβ1-treated C8-B4 microglial cell line. * P<0.05. A representative histogram as well as the mean fluorescent intensity (MFI) is shown. (B) Co-localization of pSMAD2, GFP+ and Dapi+ in the PVN obtained from a CX3CR1-GFP transgenic mouse 30 minutes after ICV injection of TGFβ1 or saline. N.D. non-detectable. Calibration bar indicates 20 μm.
Figure 5
Figure 5. Microglia mediate the effects of TGFβ1 on BP control
Systolic BP was measured by telemetry transducer in Ang II-infused CD11b-DTR mice which were treated ICV with either saline, DT, TGFβ1 or DT + TGFβ1. * P<0.05.
Figure 6
Figure 6. Molecular signature of neuroplasticity and neuroinflammation associated with hypertension and TGFβ
qRT-PCR array of PVN and brain stem lysates isolated from 4 groups of mice. Genes whose expression changed by more than twofold across all four populations are grouped into three categories: (A) inflammatory factors, (B) signaling regulators and (C) ion channels. The genes were arranged from low to high according to the fold changes in Ang II-treated mice compared with naïve controls (red column). Each column indicates the log2 value of the fold change when compared to the data from naive mice. The means of three independent experiments are shown.

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