血管紧张素II AT(1)受体通过体外自分泌转化生长因子β诱导新生大鼠心脏成纤维细胞中双糖链蛋白聚糖的表达。

Angiotensin II AT(1)-receptor induces biglycan in neonatal cardiac fibroblasts via autocrine release of TGFbeta in vitro.

作者信息

Tiede Karen, Stöter Katrin, Petrik Christian, Chen Wen Bin, Ungefroren Hendrik, Kruse Marie Luise, Stoll Monika, Unger Thomas, Fischer Jens W

机构信息

Institute of Pharmacology, Christian-Albrechts-University, Kiel, Germany.

出版信息

Cardiovasc Res. 2003 Dec 1;60(3):538-46. doi: 10.1016/j.cardiores.2003.08.009.

DOI:10.1016/j.cardiores.2003.08.009

PMID:14659799

Abstract

OBJECTIVE

After myocardial infarction, angiotensin II (AngII) promotes ventricular remodeling and deposition of extracellular matrix (ECM), e.g., collagen type 1 and 3. Whether AngII regulates the expression of small leucine-rich proteoglycans (SLRP) which are important modulators of collagen fibrillogenesis and are induced after experimental myocardial infarction in rats is not known. The aim of the present study was therefore to analyse in cultured cardiac fibroblasts the expression and secretion of the SLRP biglycan in response to AngII.

METHODS

Cardiac fibroblasts were isolated from neonatal Wistar Kyoto rats and used in the first passage. Expression of AT(1)- and AT(2)-receptors was verified by RT-PCR. Expression of protoeglycans was analyzed after metabolic labeling with [35S]-sulfate, by SDS-PAGE and Western analysis. In addition, mRNA expression was examined by means of RT-PCR and Northern analysis. The activity of the biglycan promoter was analyzed using three biglycan promoter-luciferase fusion constructs.

RESULTS

Biglycan was found to be the predominant proteoglycan produced by neonatal cardiac fibroblasts in vitro. In response to AngII (10(-7) M), secretion of total [35S]-labeled proteoglycans and mRNA of biglycan were increased to 116+/-1.8% and 121+/-11% (n=5, mean+/-S.E.M.) of unstimulated controls, respectively. Biglycan induction in response to AngII was sensitive to Losartan (10(-5) M) and unaffected by PD123177 (10(-6) M), suggesting that the AT(1)-receptor mediates the induction of biglycan. Direct activation of the biglycan promoter downstream of the AT(1)-receptor was excluded by promoter activity assays. Instead, increased release of transforming growth factor beta 1 (TGFbeta1) was detected by ELISA in response to AT(1)-receptor stimulation. Furthermore, neutralising antibodies to TGFbeta1 inhibited biglycan induction in response to AngII.

CONCLUSION

The results indicate that in cardiac fibroblasts AngII via the AT(1)-receptor causes autocrine release of TGFbeta1, which in turn induces biglycan expression and secretion.

摘要

目的

心肌梗死后，血管紧张素II（AngII）可促进心室重构及细胞外基质（ECM）如I型和III型胶原的沉积。AngII是否调节富含亮氨酸小分子蛋白聚糖（SLRP）的表达尚不清楚，而SLRP是胶原纤维形成的重要调节因子，且在大鼠实验性心肌梗死后被诱导产生。因此，本研究的目的是分析在培养的心脏成纤维细胞中，biglycan这种SLRP对AngII的反应性表达及分泌情况。

方法

从新生Wistar Kyoto大鼠分离心脏成纤维细胞并用于传代培养。通过RT-PCR验证AT(1)和AT(2)受体的表达。用[35S]-硫酸盐代谢标记后，通过SDS-PAGE和Western分析来分析蛋白聚糖的表达。此外，通过RT-PCR和Northern分析检测mRNA表达。使用三种biglycan启动子-荧光素酶融合构建体分析biglycan启动子的活性。

结果

发现biglycan是新生心脏成纤维细胞在体外产生的主要蛋白聚糖。对AngII（10(-7) M）的反应中，总[35S]标记蛋白聚糖的分泌和biglycan的mRNA分别增加到未刺激对照的116±1.8%和121±11%（n = 5，平均值±标准误）。AngII诱导的biglycan对氯沙坦（10(-5) M）敏感，而不受PD123177（10(-6) M）影响，提示AT(1)受体介导biglycan的诱导。启动子活性测定排除了AT(1)受体下游biglycan启动子的直接激活。相反，通过ELISA检测到，对AT(1)受体刺激的反应中，转化生长因子β1（TGFβ1）的释放增加。此外，TGFβ1的中和抗体抑制了对AngII的biglycan诱导。