Transforming growth factor-β1 (TGF-β1) induces pathways.with 6-15 µg of proteins were...

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Transforming growth factor-β1 (TGF-β1) induces

cerebrovascular dysfunction and astrogliosis through angiotensin II type 1 receptor-mediated signaling

pathways.

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2017-0640.R1

Manuscript Type: Article

Date Submitted by the Author: 08-Dec-2017

Complete List of Authors: Ongali, Brice; Montreal Neurological Institute and Hospital, Neurobiology; Université des Sciences de la Santé, Department of Cellular and Molecular Biology-Genetics Nicolakakis, Nektaria; Montreal Neurological Institute and Hospital, Neurobiology Tong, Xin-Kang; Montreal Neurological Institute and Hospital, Neurobiology Lecrux, Clotilde; Montreal Neurological Institute and Hospital, Neurobiology Imboden, Hans; University of Bern Hochschulstrasse 4 3012 Bern Switzerland Hamel, Edith; Montreal Neurological Institute

Keyword: losartan, enalapril, cerebrovascular reactivity, astrogliosis, TGF-β1

Is the invited manuscript for consideration in a Special

Issue?: N/A

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Transforming growth factor-β1 (TGF-β1) induces cerebrovascular

dysfunction and astrogliosis through angiotensin II type 1 receptor-

mediated signaling pathways.

Brice Ongali1†, Nicolakakis Nektaria

1, Xinkang Tong

1, Lecrux Clotilde

1, Hans

Imboden2 and Edith Hamel

1*

1Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montréal,

QC, Canada H3A 2B4; 2Institute of Cell Biology, University of Bern Baltzerstrasse 43012 Bern,

Switzerland

*Corresponding Author Address:

Edith Hamel, PhD

Montreal Neurological Institute

3801 University Street

Montréal, QC

Canada H3A 2B4

Tel: 514-398-8928

Fax: 514-398-8106

E-mail: [email protected]

†: Current address:

Department of Cellular and Molecular Biology-Genetics, Faculté de Médecine, Université des

Sciences de la Santé, Libreville, Gabon.

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Abstract

Transgenic mice constitutively overexpressing the cytokine transforming growth factor-β1

(TGF-β1) (TGF mice) display cerebrovascular alterations as seen in Alzheimer's disease (AD)

and vascular cognitive impairment and dementia (VCID), but no or only subtle cognitive

deficits. TGF-β1 may exert part of its deleterious effects through interactions with angiotensin II

(AngII) type 1 receptor (AT1R) signaling pathways. We test such interactions in the brain and

cerebral vessels of TGF mice by measuring cerebrovascular reactivity, levels of protein markers

of vascular fibrosis, nitric oxide synthase activity, astrogliosis and mnemonic performance in

mice treated (6 months) with the AT1R blocker, losartan (10 mg/kg/day), or the angiotensin

converting enzyme inhibitor enalapril (ACEi,3 mg/kg/day). Both treatments restored the severely

impaired cerebrovascular reactivity to acetylcholine, calcitonin gene-related peptide, endothelin-

1 and the baseline availability of nitric oxide in aged TGF mice. Losartan, but not enalapril,

significantly reduced astrogliosis and cerebrovascular levels of pro-fibrotic protein connective

tissue growth factor while raising levels of anti-fibrotic enzyme matrix metallopeptidase-9.

Memory was unaffected by ageing and treatments. The results suggest a pivotal role for AngII in

TGF-β1-induced cerebrovascular dysfunction and neuroinflammation through AT1R-mediated

mechanisms. Further, they suggest that AngII blockers could be appropriate against

vasculopathies and astrogliosis associated with AD and VCID.

Key words: losartan, enalapril, cerebrovascular reactivity, astrogliosis, TGF-β1, AT1 receptors.

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Introduction

Evidence suggests that the renin-angiotensin system (RAS) is altered in Alzheimer's disease

(AD) and vascular cognitive impairment and dementia (VCID) patients (Kehoe 2009), and that

angiotensin receptor blockers can lower incidence and progression to dementia (Davies et al.

2011; Li et al. 2010). The transforming growth factor beta-1 (TGF-β1), a downstream effector of

the RAS system, is a key cytokine regulator (Bye et al. 2001) that is increased in the brain and

cerebral vessels of AD and VCID patients (Grammas and Ovase 2002; Grammas et al. 2002;

Murphy et al. 2016; Trigiani and Hamel 2017; Wyss-Coray et al. 1997), and in hypertensive,

diabetic and ischemic stroke patients (Krupinski et al. 1996; Peterson 2005).

Cross-talk between TGF-β1 and the angiotensin II (AngII) type 1 receptor (AT1R) signaling

pathways have been documented (Dennler et al. 2002) in peripheral tissues and cell cultures

(Ellmers et al. 2008; Lebrethon et al. 1994; Motojima et al. 1999; Park and Han 2002). However,

it is unknown whether such cross-talk occurs in the central nervous system (CNS) of patients

suffering from AD (Grammas and Ovase 2002; Wyss-Coray et al. 1997) and VCID (Kim and

Lee 2006; Tarkowski et al. 2002). Our goal was to determine whether TGF-β1 interacts with the

AngII pathway to produce the cerebrovascular pathology and dysfunction encountered in

diseases like AD and VCID.

We used transgenic mice overexpressing a constitutively active form of TGF-β1 in astrocytes

(TGF mice). These mice exhibit cerebrovascular dysfunction (Nicolakakis et al. 2011; Wyss-

Coray et al. 2000; Zetterberg et al. 2004) including structural abnormalities associated with

increased levels of extracellular matrix (ECM) proteins, thickening of the blood vessel walls,

microvascular degenerative changes (Kalaria and Pax 1995; Tong et al. 2005; Wyss-Coray et al.

2000) and progressive functional and age-dependent deficit of cerebrovascular reactivity as well

as brain perfusion (Buckwalter et al. 2002; Gaertner et al. 2005; Tong and Hamel 2007; Tong et

al. 2005; Wyss-Coray et al. 1995). TGF mice also display activated brain immune pathways

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(Buckwalter et al. 2002; Tesseur and Wyss-Coray 2006), but intact cortical cholinergic

innervation and whisker-evoked increase in cerebral glucose uptake (Nicolakakis et al. 2011);

and memory function remains largely unaffected until late in age (Nicolakakis et al. 2011;

Papadopoulos et al. 2010; Tong and Hamel 2015). In order to discriminate possible central or

peripheral cross-talks between TGF-β1 and AngII/AT1R signalling pathways, we treated TGF

mice for 6 months (22 months at endpoint) with the brain penetrant AT1 blocker losartan or the

angiotensin converting enzyme inhibitor (ACEi), enalapril that does not penetrate the brain

(Jouquey et al. 1995). We chose these drugs, in part, because of their demonstrated benefits on

cerebrovascular function and memory in a transgenic amyloid-β overproducing mouse model of

AD (Ongali et al. 2016; Ongali et al. 2014; Royea et al. 2017).

Materials and Methods

Animals & treatments

All research protocols complied with the Guidelines of the Canadian Council on Animal Care

and were approved by the Animal Care Committee of the Montréal Neurological Institute and

McGill University. The study was conducted on different cohorts of approximately equal

numbers of male and female wild-type mice (WT) and littermate heterozygous transgenic mice

overexpressing a constitutively active form of TGF-β1 (TGF mice) under the control of the glial

fibrillary acidic protein (GFAP) promoter on a C57BL/6J background (line T64)(Wyss-Coray et

al. 1995). Transgene expression was screened by touchdown PCR using tail-extracted DNA

(Wyss-Coray et al. 1997). WT and TGF mice (body weight ~ 40 g) were treated at ̴16 months of

age for a period of 6 months (endpoint ~ 22 months). They received water, or either the selective

AT1R antagonist losartan (10 mg/kg/day, Merck Frosst Canada Ltée, Kirkland, QC, Canada) or

the ACEi enalapril maleate salt (enalapril) (3 mg/kg/day, Merck Frosst Canada Ltée, Kirkland,

QC, Canada) in the drinking water, as documented earlier (Ongali et al. 2014). In a pilot study on

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19 month-old WT and TGF mice treated for 12 weeks with the same medications, no effect was

detected on arterial blood pressure, heart rate, respiratory rate, blood gases (pO2, pCO2), and

blood pH measured according to Tong and colleagues (Tong et al. 2012) (Supplementary Table

1). Similar doses of losartan and enalapril were previously shown to be equipotent for lowering

blood-pressure (Sasaki et al. 2004), and to exert benefits on cerebrovascular or cognitive deficits

in a transgenic AD mouse model (Ongali et al. 2014). Mice were housed under a 12 h light-dark

cycle in a room with controlled temperature (25°C) and humidity (50%); food and water were

available ad libitum. In vivo tests were carried out prior to vascular reactivity studies, then tissues

were collected for subsequent protein determination and immunohistochemistry.

Learning and memory

Learning and memory were evaluated with a modified version of the Morris Water Maze

(MWM) test adapted to mice (Deipolyi et al. 2008; Papadopoulos et al. 2010). Mice underwent

first a 3 day-training with a visible platform. Then, spatial visual cues were changed, the

platform was relocated in a different quadrant of the pool and was submerged, and mice

underwent a cue trial for 5 days (3 successive trials of 90 s, separated by 45 min of rest). 24 h

after the last cue trial session (day 9), a probe trial was performed to assess memory retention

(Papadopoulos et al. 2010; Tong and Hamel 2015). Time latencies to get onto the hidden

platform, the swimming speed, as well as the time and distance traveled in the target quadrant

during the probe trial were recorded and analyzed with the 2020 Plus tracking system and Water

2020 software (Ganz FC62D video camera; HVS Image, Buckingham, UK).

Vascular and brain tissues

Mice were killed by cervical dislocation and their brains were collected in ice-cold Krebs’

solution. Middle cerebral arteries (MCA) were immediately removed under a dissecting

microscope and kept in the Krebs' solution for subsequent reactivity studies. The remaining

vessels from the circle of Willis and their ramification branches, referred to as pial vessels, were

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removed, frozen on dry ice together with cortex and hippocampus dissected from one

hemisphere, and kept at -80°C until use for further protein studies. The second hemisphere was

immersion-fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) at 4°C

(overnight), cryoprotected, frozen in isopentane (-40 °C), and stored (-80 °C) until cutting in 25

µm-thick sections with a freezing microtome, for immunohistochemistry.

Vascular reactivity

MCA segments (~ 2 mm long) were cannulated on a glass micropipette at one end and sealed to

another glass micropipette at the other end, pressurized (60 mmHg) and allowed to stabilize (~40

min) in a superfusion chamber, as previously described (Tong et al., 2005). Changes in vessel

diameter were measured using on-line video microscopy during dilatation to acetylcholine (ACh,

10-10

-10-5

M) and calcitonin gene-related peptide (CGRP, 10-10

-10-6

M) after pre-constricting the

vessels with serotonin (2×10-7

M). Constriction to endothelin-1 (ET-1, 10-10

-10-6

M) and diameter

decrease in response to nitric oxide (NO) synthase (NOS) inhibition with Nω-nitro-L-arginine (L-

NNA; 10-5

M, 35 min) were evaluated on vessels at baseline. Percent changes in vessel diameter

from basal or pre-constricted tone were plotted as a function of agonist concentration or time

course of NOS inhibition. The maximal response (EAmax) and the agonist concentration

eliciting half of the EAmax (EC50 value or pD2 (-[log EC50]) were used to determine agonist

efficacy and potency, respectively.

Protein extraction and Western blot analysis

Cerebral vessels were sonicated in Laemmli buffer, as detailed previously (Tong et al. 2005) and

protein concentration was measured by the Lowry method. Nitrocellulose membranes loaded

with 6-15 µg of proteins were pre-incubated (1 h) in blocking buffer (50 mM Tris-HCl, pH 7.5,

150 mM NaCl, 0.1% Tween 20) containing 5% skim milk, and incubated overnight with

antibodies, either rabbit anti-connective tissue growth factor (CTGF; 1:300; Abcam, Cambridge,

MA, USA), -vascular endothelial growth factor (VEGF; 1:500; Santa Cruz Biotechnology, CA,

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USA), -matrix metallopeptidase 9 (MMP9; 1:1000, Millipore, Temecula, CA, USA), -

superoxide dismutase 2 (SOD2; 1:1000; Stressgen, Victoria, BC, Canada); or goat anti-collagen

IV (Col-IV, Rockland Immunochemicals, Gilbertville, PA, USA); or mouse anti-ACE (1:500;

Abcam), -AT1R (1:500; Frei et al., 2001), -endothelial NOS (eNOS; 1:500; BD, Transduction

Laboratories, Lexington, CA, USA) or -β-actin (1:10000; Sigma-Aldrich, St.-Louis, MO). They

were then incubated (2h) with horseradish peroxidase-conjugated secondary antibodies (1:2000;

Jackson Immuno Research, West Grove, PA, USA), and proteins visualized with Enhanced

Chemi Luminescence (ECL Plus kit; Amersham Biosciences, Baie D’Urfé, QC, Canada) with a

phosphor Imager (Scanner STORM 860; GE Healthcare, NJ, USA), and quantified with Image

Quant 5.0 software (Molecular Dynamics, Sunnyvale, CA, USA).

Immunohistochemistry

Floating brain coronal sections (25µm) pretreated with H2O2 (30 min), were incubated overnight

with rabbit anti-GFAP (1:1000; DAKO, Mississauga, ON, Canada), followed by incubation for

45 mn in donkey anti-rabbit cyanin 2 (Cy2)-conjugated secondary antibody (1:400; Jackson

ImmunoResearch) for the detection of activated astrocytes. Sections were observed under the

microscope with epifluorescence (Leica, Montréal, QC, Canada), and pictures acquired with a

digital camera (Coolpix 4500; Nikon, Tokyo, Japan). Digital images (two to three sections per

mouse, three to five mice per group) taken under the same conditions were analyzed with the

MetaMorph 6.1r3 software (Universal Imaging, Downingtown, PA, USA). The areas of interest

(frontoparietal cortex) containing GFAP-positive elements were manually outlined in low-power

images. Astrogliosis was quantified in the cortex and expressed as a percent of the cortical area

occupied by GFAP-positive cells.

Statistical analysis

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Data are expressed as means ± SEM. Comparisons were analyzed by one-way analysis of

variance (ANOVA) followed by Newman-Keuls post-hoc multiple comparison tests, except for

the Morris Water Maze latencies that were analyzed by two-way ANOVA followed by

Bonferroni post-hoc multiple comparison tests. All statistical analyses were made using Graph

Pad Prism 5 software (San Diego, CA, USA) and a p value < 0.05 was considered as significant.

Results

Losartan and enalapril rescued cerebrovascular dysfunctions in aged TGF mice.

In line with our previous studies in aged TGF mice (Nicolakakis et al. 2011; Papadopoulos et al.

2010; Tong et al. 2005), ~22 month-old TGF mice exhibited reduced maximal dilatations to ACh

(-54%, p<0.001) and CGRP (-50%, p<0.01), decreases in baseline NO availability measured

after NOS inhibition (-58%, p<0.001), and lessened ET-1-mediated maximal contraction (-61%,

p<0.001) when compared to WT littermates (Fig.1, Table 1). Chronic AT1R blockade with

losartan or ACE inhibition with enalapril fully normalized cerebrovascular dilatory and

contractile function (Fig. 1). Vasomotor deficits in TGF mice and their recovery with losartan or

enalapril occurred without change in agonist potencies, ruling out the possibility of alteration in

receptor affinity and sensitivity (see Table 1).

Losartan was more potent than enalapril in altering markers of vascular fibrosis in TGF mice.

TGF mice did not display significant change in the cerebrovascular protein levels of antioxidant

enzyme SOD2 or eNOS, and neither treatment had any effect on these proteins (Fig. 2).

Angiogenic and pro-fibrotic proteins VEGF and CTGF were significantly increased in TGF

cerebrovascular tissues, whereas the increase in Col-IV did not reach statistical significance (Fig.

2). Losartan, but not enalapril, partially or fully normalized these protein levels, which was

accompanied by a concomitant significant increase in MMP-9 (Fig. 2).

Losartan, but not enalapril, attenuated astroglial activation in the brain of TGF mice.

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As previously reported in young (Lacombe et al. 2004; Nicolakakis et al. 2011; Wyss-Coray et

al. 1995) and aged (Nicolakakis et al. 2011; Tong and Hamel 2015) TGF mice, ~22 month-old

TGF mice displayed diffuse increase in GFAP-positive areas in the cerebral cortex and in the

hippocampus (Fig. 3). Diffuse GFAP-positive areas were also observed in the cortex and

hippocampus of old WT mice, but to a much lesser extent than in similarly aged TGF mice.

Losartan, but not enalapril, significantly decreased the area of activated astrocytes, as quantified

in the frontoparietal cortex of TGF mice (Fig. 3), confirming that AngII, acting through AT1R

(Nataraj et al. 1999), may be involved in the pro-inflammatory response seen in the brain of TGF

mice.

Normal cognitive performance in TGF mice is not affected by either losartan or enalapril.

Despite signs of neuroinflammation (Fig. 3) and cerebrovascular dysfunction (Fig. 1) in aged

TGF mice, these mice did not show any detectable spatial learning and memory deficits in the

MWM, and neither treatment had an impact on cognitive performance. In line with previous data

reported in 18 month-old TGF mice (Nicolakakis et al. 2011; Papadopoulos et al. 2010), the

current study suggests that 22 month-old TGF mice did not develop any memory deficit.

Additionally, cognitive performance was not affected by either treatment in WT or TGF mice

(Fig. 4).

Discussion

The substantive findings of our study are that brain TGF-β1 interacts with the AngII/AT1R

signaling cascade to induce cerebrovascular dysfunction and astrogliosis in TGF mice, and that

these AD- and VCID-related pathologies can be reversed in aged mice by drugs targeting the

RAS. Namely, we demonstrated: (1) the complete restoration of cerebrovascular reactivity by

chronic long-term inhibition of AngII effects with both losartan and enalapril in TGF mice aged

~22 months, independently from structural vascular alterations; (2) the significant reduction of

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CTGF protein levels, together with increased protein levels of MMP9; (3) the significant

reduction of astroglial activation only by losartan.

Blockade of RAS restored cerebrovascular reactivity and had beneficial effect on fibrosis

Western blot analysis (Fig. 2) and previous data (Nicolakakis et al. 2011; Tong and Hamel 2007;

Tong et al. 2005) have ruled out oxidative stress as the cause of vascular dysfunction in TGF

mice. Rather, the vascular dysfunction in these mice has been associated with altered levels

and/or activities of vasoactive proteins together with their downstream signaling cascades (Tong

and Hamel 2007, 2015; Tong et al. 2005). Vascular contractile defects to ET-1 have been

ascribed to upregulation of the ET-1 vasodilatory receptor ET-1 receptor B (ETB), and impaired

contractile ET-1 receptor A (ETA) /p38MAPK-HSP27 signaling pathway (Papadopoulos et al.

2010; Tong and Hamel 2007). Impairments in the vasodilatory responses to ACh and CGRP

could be ascribed to several factors, including reduced eNOS activity (Moilanen and Vapaatalo

1995) or protein levels (Tong and Hamel 2015), impaired endothelial TRPV4 channel signaling

(Zhang et al. 2013), dysfunction of smooth muscle cell KATP channel (Tong and Hamel 2015),

or altered state of L-type Ca2+

channels in vascular smooth muscle cells (Zhang et al. 1994).

Both losartan and enalapril restored these vasomotor responses, possibly through their actions on

these various proteins and channels (Moilanen and Vapaatalo 1995; Tong and Hamel 2015;

Zhang et al. 1994; Zhang et al. 2013) and/or through their reported ability to abrogate TGF-β1

maturation (Naito et al. 2004; Ruiz-Ortega et al. 2003; Tunon et al. 2000; Weigert et al. 2002), to

inhibit the expression of TGF-β1 receptors (Siegert et al. 1999), or to directly inactivate the

TGF-β1 Smad signaling pathway (Rodriguez-Vita et al. 2005). The latter could decrease MAPK

phosphatase 1 (MKP-1) expression involved in the inactivation of the contractile ETA

receptor/p38MAPK-HSP27 signaling pathway (Tong and Hamel 2007). Additionally, AngII

blockers have been shown to directly abolish the transcription of heat shock family genes and

normalize protein responses (Zhou et al. 2005). Altogether, these data may account for the ET-1

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contractile recovery following AngII blockers.Our findings of restored basal tone and evoked

dilatory and contractile responses, as well as the normalized levels of cerebrovascular CTGF

and VEGF levels by losartan support the involvement of AngII /AT1R signaling pathway in

cerebrovascular dysfunction induced by TGF-β1 overproduction in the brain of TGF mice, and

formally suggest interactions between AngII/AT1R and TGF-β1 (Perbal 2004; Ruiz-Ortega et al.

2007; Ruperez et al. 2003; Wang et al. 2004). The results with enalapril point to a peripheral site

of action, possibly at the level of the endothelial cells since enalapril does not act centrally

(Jouquey et al. 1995).

We did not find significant change in the protein levels of Col-IV, previously reported to be

increased in younger TGF mice (Nicolakakis et al. 2011; Tong et al. 2005). It could be that

normal aging increases other makers of fibrosis in TGF mice, such as fibronectin and versican,

which are coincidentally elevated in the thickened tunica intima of VCID patients suffering from

cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy

(CARASIL) (Lan et al. 2013) and of hypertensive rats (SHR) (Ando et al. 2004; Che et al. 2008).

It could also be due by the increase in MMP9 induced by losartan that led to reduced Col-IV, as

MMP9 has collagenolytic activity (Newby et al. 2006) that may promote fibrosis and

calcification of the ECM (Perrotta et al. 2016; Yabluchanskiy et al. 2013)

Taken altogether, these data not only support the antifibrotic effects of RAS blockers (Park et al.

2012), but further indicate that with aging there are ECM modulations that may contribute to the

altered structure of the blood vessels (Newby et al. 2006; Perrotta et al. 2016). Importantly, our

data confirmed the possibility of rescuing cerebrovascular function even when the structure of

the vessels remains deeply altered (Nicolakakis et al. 2011; Papadopoulos et al. 2010; Tong et al.

2005).

Central effects of losartan, but not of enalapril.

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Aged TGF mice displayed extensive astrocyte activation, and this was attenuated by losartan.

This is in keeping with the fact that astrocytes are the major source of angiotensinogen in the

brain (Kandalam and Clark 2010), and together with microglia and neurons, express

AT1R(Grammas and Ovase 2002; Lanz et al. 2010; Muller et al. 2000; Ruiz-Ortega et al. 1998).

(Li et al. 1993) Enalapril failed to reduce astrocyte activation, likely resulting from its incapacity

to penetrate the brain parenchyma (Jouquey et al. 1995). We previously showed that losartan also

significantly reduced astroglial activation in the brain of an AD mouse model, based on its ability

to cross the BBB, (Ongali et al. 2014; Royea et al. 2017),likely via angiotensin IV (AngIV)-

mediated mechanisms, and that similar to the present findings, enalapril failed to do so (Ongali et

al. 2016). Therefore, our data suggest that blocking AngII/AT1R signaling could represent a

therapeutic avenue against astrogliosis and/or neuroinflammation for patients suffering from AD,

VCID, hypertension and small vascular diseases (SVD), in which increases in TGF-β1 are part of

the pathology (Frances et al. 2016; Ho and Yeh 2017; Mogi et al. 2009).

Cognitive function in aged TGF mice.

We did not find any learning or memory deficits in 22 month-old TGF mice, extending our

previous work on 18 month-old TGF mice (Nicolakakis et al. 2011; Papadopoulos et al. 2010),

and emphasizing that cerebrovascular dysfunction alone, or at least at the level seen in aged TGF

mice, is insufficient to impair memory.

TGF mice can be viewed as a model of chronic cerebrovascular deficiency (Gaertner et al. 2005;

Nicolakakis et al. 2011; Tong and Hamel 2015) that recapitulates well the fibrotic pathology

seen in AD, VCID and CARASIL (Lan et al. 2013; Tikka et al. 2014), which can be used for

future research.

Interaction between TGF-β1 and AngII/AT1R signaling

TGF-β1 is among the downstream components of the AngII/ATR1 signaling pathways

(Grammas and Ovase 2002; Wyss-Coray et al. 2000), but our results in TGF mice

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overexpressing biologically active TGF-β1 suggest that TGF-β1 may in turn act upon the

AngII/ATR1 signaling pathway and result in the cerebrovascular and neuroinflammatory

pathologies observed in these mice. Consistent with this observation, various interaction loops

between TGF-β1 and the RAS have been documented in peripheral tissues and cell cultures

(Ellmers et al. 2008; Lebrethon et al. 1994; Motojima et al. 1999; Park and Han 2002). For

example, increases in AngII binding sites and AT1R have been found in response to TGF-β1 in

human adrenal cells (Lebrethon et al. 1994), and attributed to cross-talks between AT1Rs and

specific kinase signaling pathways that are simultaneously activated by TGF-β1 (Dennler et al.

2002). .In mice lacking the TGF-β1 gene, AngII fails to induce fibrosis or hypertrophy (Schultz

Jel et al. 2002). Similar interactions between TGF-β1 and AngII/AT1R-mediated signaling

pathways may occur in AD or VCID vessels or the normal brain. Additional studies are needed

to investigate whether the AngIV/AT4R signaling cascade, which mediates the cerebrovascular

and neuronal benefits of losartan in a mouse model of AD (Ongali et al. 2014; Royea et al.

2017), also plays a role in losartan benefits in TGF mice.

Conclusions

We conclude that AngII brain penetrant blockers could be considered as a therapeutic approach

against TGF-β1-mediated vascular reactivity impairment and neuroinflammation. Our data also

support for the first time, the possibility that TGF-β1 may partly exert its detrimental effects

through AngII/AT1R-mediated signaling pathways in the brain and cerebral vessels of TGF

mice. This suggests the possibility that TGF-β1 interacts with AngII/AT1R-mediated signaling

pathways involved in AD- and VCID-associated neuroinflammation and cerebrovasculopathies,

which should be taken in account for further studies and therapies.

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Acknowledgements

Supported by research grants from the Alzheimer Society of Canada and the the Canadian

Institutes of Health Research (CIHR, MOP-84275 and MOP-126001) (EH), Les Fonds de la

Recherche en Santé du Québec and Jeanne Timmins Fellowships (BO). Authors thank Dr L.

Mucke (Gladstone Inst of Neurological Disease and Dept. Neurology, UCSF, CA, USA) for the

TGF-β1 transgenic mouse breeders and Merck Research Laboratories for their generous supply

of losartan and enalapril. Nella Serlucca is also acknowledged for her technical assistance in

reactivity.

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Table 1. Effects of losartan (L) or enalapril (E) on cerebrovascular responses in aged TGF mice.

Losartan (L) and Enalapril (E) both rescued impaired cerebrovascular reactivity to ACh, CGRP,

ET-1 and NOS inhibition by L-NNA in TGF treated mice. Data are means ± SEM of the number

(n) of animals in parentheses, and represent the best fitted values of maximal agonist response

(EAmax) or potency (pD2, -[logEC50]). EAmax is the percent maximal dilatation to ACh and

CGRP, constriction to ET-1 or percent maximal diameter decrease after 35 min inhibition of

NOS with 10-5

M L-NNA. WT: Wild-type; TGF: Transgenic mice overexpressing transforming

growth factor-β1. ��,*** p<0.001. �: WT vs. TGF mice, *: treated vs. untreated TGF mice

using one-way ANOVA, followed by Newman-Keuls post-hoc test.

WT

(n=4)

WT(L)

(n=3)

WT(E)

(n=4)

TGF

(n=5)

TGF(L)

(n=6)

TGF(E)

(n=5)

ACh EAmax 65.1 ± 2.0 58.0 ± 2.0 51.1 ± 2.8 29.3±1.7��, *** 53.7 ± 1.5 54.7 ± 2.4

pD2 7.59 ± 0.11 7.79 ± 0.12 7.59 ± 0.19 7.36 ± 0.17 7.82 ± 0.09 7.71 ± 0.15

CGRP EAmax 57.3 ± 3.8 55.1 ± 1.7 42.6 ± 2.0 28.9±2.2��, *** 52.8 ± 1.9 45.9 ± 3.2

pD2 8.40 ± 0.23 8.45 ± 0.12 8.38 ± 0.15 8.07 ± 0.23 8.53 ± 0.14 8.27 ± 0.24

ET-1 EAmax 54.8 ± 2.4 67.5 ± 4.2 54.6 ± 2.3 21.6±1.4��, *** 56.0 ± 1.1 52.4 ± 2.2

pD2 8.14 ± 0.13 8.64 ± 0.26 8.18 ± 1.13 8.20 ± 0.20 8.68 ± 0.08 8.56 ± 0.15

LNNA EAmax 54.7 ± 0.7 69.1 ± 3.5 57.2 ± 2.2 28.3±2.0��, *** 64.1± 4.1 56.8 ± 0.7

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Figure Legends

Figure 1. Altered cerebrovascular reactivity in aged TGF mice.

Compared to WT mice (�), age-matched TGF mice (�) displayed impaired cerebrovascular dilatory

responses to acetylcholine (ACh) and calcitonin gene-related peptide (CGRP), reduced contractile

responses to endothelin-1 (ET-1) and in baseline NO bioavailability tested by inhibiting NOS with 10-

5M L-NNA (arrow indicates time of L-NNA application). Whereas treatment with losartan (L) or

enalapril (E) had no effects on WT () cerebrovascular reactivity, these treatments fully rescued

dilatory response to ACh, CGRP and contractility to ET-1 and NOS inhibition in treated TGF mice (�).

Data are Mean ± SEM. Comparisons to WT (�) or to TGF (*) are indicated by�,* p<0.05; ��, ** p <

0.01 and ��,*** p < 0.001, by one-way ANOVA followed by a Newman Keuls post-hoc test.

Figure 2: Cerebral blood vessels from aged TGF mice display fibrosis and alterations in vascular

signaling. Levels of proteins involved in oxidative stress (SOD2), the synthesis of NO (eNOS), vascular

structure (Col-IV), fibrosis/remodeling (MMP9, CTGF, VEGF), and AT1Rs were measured in TGF

mouse (�) pial vessels. Only CTGF and VEGF were significantly increased in TGF mice compared to

WT controls. Losartan (L, �) but not enalapril (E, �) counteracted these increases (albeit not

significantly for VEGF) and significantly increased levels of MMP9. (n=4 mice/group). Data are Mean

± SEM. Comparisons to WT (�) or to TGF (*) are indicated by�,* p<0.05; ��, ** p< 0.01 by one-

way ANOVA followed by a Newman Keuls post-hoc test.

Figure 3: Astroglial activation in the brain of aged TGF mice.

Enhanced astrocyte activation seen as diffuse GFAP-positive material in the brains of aged TGF mice

contrasted with smaller activation detected in the cortex (Co) or hippocampus (Hi) of wild-type (WT)

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littermates. When quantified in the frontoparietal cortex (outlined area in WT panel), losartan (L), but

not enalapril (E, �) treatment significantly lowers the density of activated astrocytes in TGF-treated

mice. Scale bar, 20µm. (n=4 mice/group). Error bars represent SEM. �� p<0.01; ***��p<0.001

when compared with WT (�) or TGF (*) mice using one-way ANOVA followed by a Newman Keuls

post-hoc test.

Figure 4: Aged TGF mice did not display spatial learning and memory deficits. Aged TGF mice

(�) performed as well as their WT littermate controls (�) in both spatial learning and spatial memory

(probe trial) in the Morris Water Maze. Losartan (L, �) or enalapril (E, �) had no effect on learning

and memory of TGF mice. Mice did not have any motor or visual deficits as they all rapidly found the

visible platform (days 1-3). Error bars represent SEM (n=7-16 mice/group).

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