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. 2013 May 9:5:19.
doi: 10.3389/fnagi.2013.00019. eCollection 2013.

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

Debjani Tripathy  1 , Alma Sanchez, Xiangling Yin, Jinhua Luo, Joseph Martinez, Paula Grammas
Affiliations

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

Debjani Tripathy et al. Front Aging Neurosci. .

Abstract

Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01-0.001) increases in thrombin, HIF-1α, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1α, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01-0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.

Keywords: Alzheimer's disease; dabigatran; endothelial cells; hypoxia; neuroinflammation; thrombin.

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Figures

Figure 1
Figure 1
Brain microvascular endothelial cell cultures were treated with 1 nM direct thrombin inhibitor, dabigatran (DTI) and were exposed to hypoxia for 6 h. Total RNA was collected, reverse transcribed, and mRNA expression of HIF-1α, thrombin, IL-6, MCP-1, and MMP2 determined by real-time PCR. Data are from four separate experiments performed in triplicate and expressed as fold change over control (C). ***p < 0.001 vs. C (control); ap < 0.001, bp < 0.01 vs. H (hypoxia).
Figure 2
Figure 2
Brain tissues from control, control + DTI, AD and AD + DTI AD mice were fixed and immunostained with HIF-1α, thrombin, IL-6, MCP-1, and MMP2 primary antibodies and fluorescence labeled secondary antibody (green). The bar graph denotes signal intensities normalized to endothelial specific marker von Willebrand factor (vWF, red) and control values set to 1. Data are from four mice per group. *p < 0.05, ***p < 0.001 vs. C (control); ap < 0.001, cp < 0.05 vs. AD.
Figure 3
Figure 3
Brain tissues from control, control + DTI, AD and AD + DTI mice were homogenized, total RNA collected, reverse transcribed and mRNA expression of HIF-1α, thrombin, IL-6, MCP-1, and MMP2 determined by real time PCR. Data are from four mice per group and expressed as fold change over control (C, untreated). ***p < 0.001 vs. C (control); ap < 0.001, bp < 0.01 vs. AD.
Figure 4
Figure 4
Brain tissue sections from frontal cortices from control, control + DTI, AD, and AD + DTI mice were incubated for 30 min with 5 μM of Dihydroethidium (DHE, red) fluorescence dye, and NucBlue stain (blue). Data represents signal intensities of DHE stained cells to non-stained cells. ***p < 0.001 vs. control (C); ap < 0.001 vs. AD.
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