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Ethanol Gestational Exposure Impairs Vascular Development and Endothelial Potential to Control BBB-Associated Astrocyte Function in the Developing Cerebral Cortex

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Abstract

Ethanol consumption during pregnancy or lactation period can induce permanent damage to the development of the central nervous system (CNS), resulting in fetal alcohol spectrum disorders (FASD). CNS development depends on proper neural cells and blood vessel (BV) development and blood-brain barrier (BBB) establishment; however, little is known about how ethanol affects these events. Here, we investigated the impact of ethanol exposure to endothelial cells (ECs) function and to ECs interaction with astrocytes in the context of BBB establishment. Cerebral cortex of newborn mice exposed in utero to ethanol (FASD model) presented a hypervascularized phenotype, revealed by augmented vessel density, length, and branch points. Further, aberrant distribution of the tight junction ZO-1 protein along BVs and increased rates of perivascular astrocytic endfeet around BVs were observed. In vitro exposure of human brain microcapillary ECs (HBMEC) to ethanol significantly disrupted ZO-1 distribution, decreased Claudin-5 and GLUT-1 expression and impaired glucose uptake, and increased nitric oxide secretion. These events were accompanied by upregulation of angiogenesis-related secreted proteins by ECs in response to ethanol exposure. Treatment of cortical astrocytes with conditioned medium (CM) from ethanol exposed ECs, upregulated astrocyte’s expression of GFAP, Cx43, and Lipocalin-2 genes, as well as the pro-inflammatory genes, IL-1beta, IL-6, and TNF-alpha, which was accompanied by NF-kappa B protein nuclear accumulation. Our findings suggest that ethanol triggers a dysfunctional phenotype in brain ECs, leading to impairment of cortical vascular network formation, and promotes ECs-induced astrocyte dysfunction, which could dramatically affect BBB establishment in the developing brain.

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Data Availability

All data generated and analyzed during this study are available from the corresponding author upon reasonable request.

Abbreviations

FASD:

Fetal alcohol spectrum disorder

ECs:

Endothelial cells

CNS:

Central nervous system

ZO:

Zonula occludens

CM:

Conditioned medium

BBB:

Blood-brain barrier

BV:

Blood vessel

GLUT:

Glucose transporter

HBMEC:

Human brain microcapillary endothelial cell

MBEC:

Mouse brain endothelial cell

EC-CM:

Endothelial cell-conditioned medium

TiJOR:

Tight junction organization rate

GFAP:

Glial fibrillary acidic protein

MTT:

3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide

2-NBDG:

2-(N-(7-Nitrobenz-2-oxa-1,3- diazol-4-yl)amino)-2-deoxyglucose

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Acknowledgments

We thank Dr. Ana Paula Cabral de Araújo Lima (Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Brazil) that with the consent of Dr. Dennis Grab (Associate Professor, Johns Hopkins University, Department of Pathology, Baltimore, USA) provided HBMEC cell line. We also thank Marcelo Meloni, Carolina Moraes, and Grasiela Ventura for technical assistance.

Funding

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (MS, JS, FG); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (MS, JS, FCAG); Departamento de Ciência e Tecnologia do Ministério da Saúde (Decit) (APBA); Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (MS, JS, FCAG); and International Society for Neurochemistry (ISN) (JS).

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  1. Laboratório de Neurobiologia Celular, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Bloco F, Sala F15, Ilha do Fundão, Rio de Janeiro, RJ, 21949-902, Brazil

    Michele Siqueira, Ana Paula Bérgamo Araujo, Flávia Carvalho Alcantara Gomes & Joice Stipursky

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  1. Michele Siqueira
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  2. Ana Paula Bérgamo Araujo
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  3. Flávia Carvalho Alcantara Gomes
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Contributions

Conceptualization, JS; methodology, JS and MS; formal analysis and investigation, APBA and MS; visualization, MS; writing (original draft), MS; writing (review and editing), FCAG and JS; funding acquisition, FCAG and JS; resources, FCAG and JS; project administration, JS; supervision, JS

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Correspondence to Joice Stipursky.

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All animal protocols were approved by the Animal Use Ethics Committee of the Federal University of Rio de Janeiro (protocol n°. CEUA 067/16).

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Supplementary Information

Supplementary figure 1

Treatment of astrocytes with ethanol modulates BBB and pro-inflammatory related genes in a less extent than ECs derived factors. Confluent cortical astrocytes cultures were treated with control medium (Control) or ethanol for 24 h, followed by RT-qPCR analysis. Ethanol significatively enhanced Aqp4, Cx43, GFAP and TNF-α and did not modulate Lcn2, IL-1β and IL-6 gene expression (a-b). Immunocytochemistry assays revealed that ethanol exposure did not induce NF-κB nuclear accumulation in astrocyte cultures (b). Binary masks of nuclear and cytoplasmic labeling intensities of NF-κB ROI (g-j). Data are expressed as Mean ± SEM (n = 3–4 control and ethanol-treated) independent astrocyte cultures. Scale bar: 20 μm (c-j). (PNG 460 kb)

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Siqueira, M., Araujo, A.P.B., Gomes, F.C.A. et al. Ethanol Gestational Exposure Impairs Vascular Development and Endothelial Potential to Control BBB-Associated Astrocyte Function in the Developing Cerebral Cortex. Mol Neurobiol 58, 1755–1768 (2021). https://doi.org/10.1007/s12035-020-02214-8

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  • DOI: https://doi.org/10.1007/s12035-020-02214-8

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