Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 390, Issue 11, pp 1135–1144 | Cite as

T3 peptide, a fragment of tumstatin, stimulates proliferation and migration of cardiac fibroblasts through activation of Akt signaling pathway

  • Jumpei Yasuda
  • Kana Fukui
  • Muneyoshi Okada
  • Hideyuki Yamawaki
Original Article
First Online:
Received:
Accepted:

Abstract

Proliferation and migration of cardiac fibroblasts are important in early stage of wound-healing after myocardial infarction. The effects of tumstatin, a cleaved fragment of collagen type IV α3 chain, on these functions of cardiac fibroblasts have not been clarified. In this study, we examined it by using T3 peptide, an active fragment of tumstatin. Cardiac fibroblasts were isolated from ventricles of adult male Wistar rats. Proliferation was examined by a cell counting assay. Boyden chamber assay was performed to examine migration. Expression and phosphorylation of proteins were determined by Western blotting. T3 peptide (300 ng/ml, 24 h) significantly increased proliferation and migration of cardiac fibroblasts. T3 peptide (300 ng/ml, 30 min) significantly increased Akt (Ser473) phosphorylation. LY294002 (10 μM, 30 min pretreatment), a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, significantly inhibited the T3 peptide-induced proliferation, migration, and activation of Akt signaling pathway in cardiac fibroblasts. Cilengitide, an inhibitor of integrin αvβ3/αvβ5, suppressed Akt phosphorylation and proliferation of cardiac fibroblasts. Expression of tumstatin decreased in the infarcted area of rat model of myocardial infarction. We for the first time demonstrated that T3 peptide stimulates proliferation and migration at least partly through the activation of PI3K/Akt signaling pathway via binding integrin αvβ3/αvβ5 in adult rat cardiac fibroblasts. These results indicate that tumstatin promotes the initial stage of wound-healing through activation of cardiac fibroblasts after myocardial infarction.

Keywords

Cardiac fibroblasts Migration Proliferation Tumstatin Wound-healing 

Abbreviations

CC8

Cell counting kit-8

DMEM

Dulbecco’s modified Eagle’s medium

ECM

Extracellular matrix

ERK

Extracellular signal-regulated kinase

FAK

Focal adhesion kinase

FBS

Fetal bovine serum

IL

Interleukin

JNK

c-jun N-terminal kinase

MMPs

Matrix metalloproteinases

mTOR

Mammalian target of rapamycin

PDK1

Phosphoinositide-dependent protein kinase 1

PI3K

Phosphoinositide 3-kinase

S.E.M.

Standard error of the mean

TBS

Tris-buffered saline

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Notes

Acknowledgments

This study was supported by Grant-in-Aid for Japan Society for the Promotion of Science Research Fellow Grant Number (JP16J07684) and School of Veterinary Medicine, The Kitasato University.

Compliance with ethical standards

All animal experiments were approved by the President of Kitasato University through the judgment by Institutional Animal Care and Use Committee of Kitasato University (Approval no. 15-047, 16-032 and 16-057). Adult male Wistar rats (4–10 weeks old, CLEA Japan, Tokyo, Japan) were cared in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines for animal care and treatment of the Kitasato University.

Supplementary material

210_2017_1413_MOESM1_ESM.docx (59 kb)
Supplemental Figure S1 (DOCX 59 kb)
210_2017_1413_MOESM2_ESM.docx (694 kb)
Supplemental Figure S2 (DOCX 694 kb)
210_2017_1413_MOESM3_ESM.docx (39 kb)
Supplemental Figure S3 (DOCX 39 kb)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Laboratory of Veterinary Pharmacology, School of Veterinary MedicineKitasato UniversityTowadaJapan

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