Losartan prevents acquired epilepsy via TGF-β signaling suppression

Abstract

Objective: Acquired epilepsy is frequently associated with structural lesions after trauma, stroke, and infections. Although seizures are often difficult to treat, there is no clinically applicable strategy to prevent the development of epilepsy in patients at risk. We have recently shown that vascular injury is associated with activation of albumin-mediated transforming growth factor β (TGF-β) signaling, and followed by local inflammatory response and epileptiform activity ex vivo. Here we investigated albumin-mediated TGF-β signaling and tested the efficacy of blocking the TGF-β pathway in preventing epilepsy.

Methods: We addressed the role of TGF-β signaling in epileptogenesis in 2 different rat models of vascular injury, combining in vitro and in vivo biochemical assays, gene expression, and magnetic resonance and direct optical imaging for blood-brain barrier permeability and vascular reactivity. Long-term electrocorticographic recordings were acquired in freely behaving animals.

Results: We demonstrate that serum-derived albumin preferentially induces activation of the activin receptor-like kinase 5 pathway of TGF-β receptor I in astrocytes. We further show that the angiotensin II type 1 receptor antagonist, losartan, previously identified as a blocker of peripheral TGF-β signaling, effectively blocks albumin-induced TGF-β activation in the brain. Most importantly, losartan prevents the development of delayed recurrent spontaneous seizures, an effect that persists weeks after drug withdrawal.

Interpretation: TGF-β signaling, activated in astrocytes by serum-derived albumin, is involved in epileptogenesis. We propose losartan, a drug approved by the US Food and Drug Administration, as an efficient antiepileptogenic therapy for epilepsy associated with vascular injury.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

© 2014 American Neurological Association.

Figures

Figure 1

Albumin uptake into astrocytes is associated with TGF-β1 up-regulation. (A) Fluorescent imaging demonstrating albumin uptake into a cultured astrocyte. (B) Albumin uptake dynamics shows significant uptake by cultured astrocytes (Ast, filled circles) and not by neurons (Neu, open circles). (C) Uptake of albumin (Alb) (24 hours) by astrocytes is significantly blocked in the presence of the specific TGF-βR blocker, SB431542 (SB) or the caveolae-mediated endocytosis blocker methyl-β-cyclodextran (MBCD). (D) Western blot analysis revealing significantly higher levels of ALK5 in astrocytic cultures compared to neurons. (E) Real-time PCR and (F) ELISA, showing albumin-induced up-regulation of TGF-β1 mRNA and protein secretion from astrocytes, but not from neurons. Albumin-induced TGF-β1 up-regulation was blocked by the TGF-βRs antagonists SB431542 (SB) or SJN2511 (SJN). *p≤0.05.

Figure 2

Exposure of astrocytes to albumin is associated with Smad2/3 phosphorylation. Analysis of p-Smad1/5/8 and p-Smad2/3 levels 4 and 24 hours post exposure to albumin (Alb) in neurons (A) and astrocytes (B), reveals a robust increase in astrocytic p-Smad2/3 4 hours after treatment. The ALK1 and ALK5 receptor agonists, Bmp6 and TGF-β1, served as positive controls. (C) A time course experiment demonstrating a gradual increase in p-Smad2/3 levels following exposure to albumin prevented using the TGF-βR antagonists, SB431542 (SB) or SJN2511 (SJN). Note that fatty acid free albumin (Pure alb) still elicited an increase in p-Smad2/3 levels. *p≤0.05.

Figure 3

Losartan prevents albumin-induced brain TGF-β signaling in vivo. (A) Losartan (Los) significantly blocks albumin-induced (Alb) increase in p-Smad2/3 levels in vivo (48 hours) and (B) prevents the increase in GFAP mRNA levels, commonly considered as a marker for astrogliosis, 7 days after treatment. (C) In vivo surface imaging shows no effect of losartan or albumin on arterial diameter, while Bradykinin (BK) induced significant vasodilation compared to control (ACSF). (D) Blood flow curves, calculated from fluorescent angiography, showing no effect of losartan (Los) on regional cerebral blood flow compared to ACSF (Ctrl). (E) Cortical vessels under ACSF (Ctrl) and increased arterial diameter (arrows) upon perfusion with Bradykinin (BK). *p≤0.05.

Figure 4

Losartan prevents albumin-induced epilepsy. (A) Treatment protocol; animals were treated with local application of albumin (Alb) in the presence or absence of losartan (Los), added to the ACSF. (B) In-vivo imaging of cortical vessels during local application of fluorescently labeled albumin (green) and Evans blue (EB, red) intravenous injection, indicating no EB extravasation, consistent with normally functioning BBB. (C) The development of epilepsy was assessed through the occurrence of spontaneous electrocorticographic seizures (see exemplary seizure of an albumin-treated rat). (D) Color coded table showing the number of seizures per day is presented for each rat in albumin- (Alb) compared to ACSF- (Ctrl) and losartan- (Los) treated animals (see scale bar). (E) Bar graphs represent the percent of epileptic animals, number of seizures per week and the duration of seizures. Overall, compared to the albumin group, losartan treatment significantly lowered the percent of epileptic animals, and the number and duration of seizures among animals that did develop epilepsy *p ≤ 0.05.

Figure 5

Losartan prevents epilepsy following BBB dysfunction. (A) Treatment protocol; animals were treated with local application of DOC in the presence or absence of losartan. A single dose of losartan was injected (100mg/kg) at the end of the surgical procedure, followed by 3 weeks of oral administration (2gr/L in the drinking water). (B) In-vivo imaging of cortical vessels during local application of DOC shows BBB opening and leakage of Evans blue-albumin complexes. (C) T2 sequence brain MRI 24 hours following DOC shows edema in the treated area (arrow). A T1 image shows areas of significantly increased signal following gadolinium-DTPA injection (color bar represents percentage of contrast enhancement). (D) Color coded table showing the number of seizures per day is presented for each rat in DOC- compared to ACSF- (Ctrl) and losartan- (Los) treated animals (see scale bar). (E) Bar graphs represent the percent of epileptic animals, number of seizures per week and the duration of seizures. Overall, compared to the DOC group, losartan treatment significantly lowered the percent of epileptic animals and the number of seizures among animals that did develop epilepsy *p ≤ 0.05.

Figure 6

A schematic diagram illustrating that under dysfunctional BBB the extravasation of albumin into the brain’s extracellular space leads to its interaction with astrocytic TGF-βRs, activation of TGF-β signaling and secretion of TGF-β1, followed by neuronal hyperexcitability and un-provoked epileptic seizures.