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Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension

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Abstract

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

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Abbreviations

ADO:

Adenosine

ADP:

Adenosine 5′-diphosphate

AMP:

Adenosine 5′-monophosphate

ATP:

Adenosine 5′-triphosphate

AngII:

Angiotensin II

DOCA:

Deoxycorticosterone acetate

EC:

Endothelial cell

IL:

Interleukin

IFN-γ:

Interferon gamma

MRA:

Mesenteric resistance artery

SHR:

Spontaneously hypertensive rat

NTPDase:

Nucleoside triphosphate diphosphohydrolase

RT-qPCR:

Reverse transcription quantitative real-time PCR

TGF-β1:

Transforming growth factor beta 1

TNF-α:

Tumor necrosis factor alpha

VSMC:

Vascular smooth muscle cell

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Acknowledgments

The authors thank Dimitri Bréard for expert HPLC technical assistance.

JS received support from the Canadian Institutes of Health Research (CIHR) and was also the recipient of a “Chercheur National” Scholarship from the Fonds de Recherche du Québec – Santé (FRQS).

Funding

MitoVasc Institute was supported by INSERM, CNRS, University of Angers, CHU of Angers, Région Pays de la Loire, Angers-Loire Métropole, and Département du Maine et Loire. CR was supported by grants from INSERM and Région Pays de la Loire.

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Authors and Affiliations

  1. MITOVASC Institute - UMR CNRS 6015 INSERM U1083 University of Angers, Angers, France

    Charlotte Roy, Antoine Caillon, Julie Favre, Anne Laure Guihot, Ludovic Martin, Daniel Henrion & Gilles Kauffenstein

  2. CNRS UMR 6299, INSERM 892, CRCNA, University of Angers, Angers, France

    Julie Tabiasco & Yves Delneste

  3. University Hospital of Angers, Angers, France

    Yves Delneste, Ludovic Martin, Daniel Henrion & Gilles Kauffenstein

  4. L’institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes, France

    Jean Merot

  5. CNRS, UMR 7355, Orleans, France

    Daniele C. Nascimento & Bernhard Ryffel

  6. CNRS UMR 7355, INEM, University of Orleans, Orleans, France

    Daniele C. Nascimento & Bernhard Ryffel

  7. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Harvard University, Boston, MA, USA

    Simon C. Robson

  8. Département de microbiologie-infectiologie et d’immunologie, Faculté de Médecine, Université Laval, Québec City, QC, G1V 0A6, Canada

    Jean Sévigny

  9. Centre de recherche du CHU de Québec – Université Laval, Québec City, QC, G1V 4G2, Canada

    Jean Sévigny

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  1. Charlotte Roy
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Corresponding author

Correspondence to Gilles Kauffenstein.

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Conflicts of interest

Charlotte Roy declares that she has no conflict of interest.

Julie Tabiasco declares that she has no conflict of interest.

Antoine Caillon declares that he has no conflict of interest.

Yves Delneste declares that he has no conflict of interest.

Jean Merot declares that he has no conflict of interest.

Julie Favre declares that she has no conflict of interest.

Anne Laure Guihot declares that she has no conflict of interest.

Ludovic Martin declares that he has no conflict of interest.

Daniele Nascimento declares that she has no conflict of interest.

Bernhard Ryffel declares that he has no conflict of interest.

Simon C. Robson declares that he has no conflict of interest.

Jean Sévigny declares that he has no conflict of interest.

Daniel Henrion declares that he has no conflict of interest.

Gilles Kauffenstein declares that he has no conflict of interest.

Ethical approval

Animals were manipulated in accordance with European Community Standards on the Care and Use of Laboratory Animals (authorization No. 6422).

Electronic supplementary material

Fig. S1

Arterial expression of NTPDase family members as an effect of hypertension. NTPDase 1, 2, 3, and 8 mRNA expression was determined in thoracic aorta of sham and AngII-infused mice (n = 5) by RT-qPCR. Data are presented as means ± SEM. **p < 0.01 (Student’s t-test) vs. controls. (PDF 43.9 kb)

Fig. S2

ADPase activity in mouse abdominal aortic homogenates. ADPase activity was measured by HPLC with fluorescence etheno-ADP as a substrate as described in methods. Entpd1+/− mice display a 45% reduction in ADPase activity compared to wild-type (1.82 ± 0.2 vs. 3.3 ± 0.3 nmol/μg protein/h). More than 90% ADPase activity is lost in Entpd1−/− mice compared to wild-type activity (0.24 ± 0.02 vs. 3.3 ± 0.3 nmol/μg/h). These results confirmed a 50% reduction in CD39 expression in Entpd1+/− animals. Data are presented as means ± SEM (n = 4–6 per group). ***p < 0.001 (Student’s t-test) vs. wild-type aorta homogenate. (PDF 212 kb)

Fig. S3

Orbital shear stress induces upregulation of CD39 mRNA expression in vitro. Endothelial cells (three independent wells) were exposed to orbital shear stress (210 rpm = 11.5 dynes/cm2) for 24 h by using an orbital shaker as previously described (Dardik et al. J. Vasc. Surg 2005). CD39 mRNA expression was normalized to the reference gene and compared to cells cultured in static condition. Data represent the mean ± SEM. **p < 0.01 (Student’s t-test) vs. control cells. (PDF 37.1 kb)

Table S1

Source and concentration of molecules used to stimulate vascular cells (PDF 173 kb)

Table S2

Sequences of primer pairs used for RT-qPCR (PDF 118 kb)

Table S3

Gene expression variation evaluated by RT-qPCR in ECs and VSMCs submitted to cyclic stretch (15%, 0.5 Hz). Data are presented as means ± SEM of five independent VSMC cultures and three (for 72 h) or five (for 6, 24 h) independent EC cultures. * p < 0.05 (Student’s t-test) vs. time-matched control. (PDF 116 kb)

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Roy, C., Tabiasco, J., Caillon, A. et al. Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension. Purinergic Signalling 14, 73–82 (2018). https://doi.org/10.1007/s11302-017-9597-9

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