Elsevier

Phytomedicine

Volume 19, Issue 1, 15 December 2011, Pages 13-19
Phytomedicine

Matrix metalloproteinase-9 induces cardiac fibroblast migration, collagen and cytokine secretion: Inhibition by salvianolic acid B from Salvia miltiorrhiza

https://doi.org/10.1016/j.phymed.2011.06.024Get rights and content

Abstract

Cardiac fibroblasts play the key role in cardiac function and matrix metalloproteinases-9 (MMP-9) is a well known contributor to the development of myocardial remodeling. However, the direct regulation of MMP-9 on the function of cardiac fibroblasts and the underlying mechanism are far from elucidation.

In the present research, recombinant protein encoding catalytic domain of MMP-9 (MMP-9 CD) was constructed and the function of neonatal cardiac fibroblasts was investigated by cell proliferation assay, migration assay, picrosirius red assay, multiplex cytokine assay and fibroblast phenotype detection. 200 nM MMP-9 CD stimulated cardiac fibroblasts migration (169.4 ± 22.5% versus 100 ± 0%, p < 0.01), increased collagen synthesis (1.5 ± 0.2 fold, p < 0.05), up-regulated the secretion of ICAM (574.0 ± 40.1 versus 268.5 ± 8.6 pg/ml, p < 0.01), TNF-α (192.6 ± 11.0 versus 14.4 ± 1.8 pg/ml, p < 0.001), IL-6 (1500.9 ± 70.2 versus 323.4 ± 40.6 pg/ml, p < 0.001) and sVCAM-1 (30.3 ± 4.3 versus 7.0 ± 0.1 pg/ml, p < 0.05) and down-regulated VEGF (436.5 ± 148.9 versus 1034.3 ± 28.1 pg/ml, p < 0.05) significantly with modest effects on proliferation. Accompanying with these regulations, transition of fibroblasts to myofibroblast was confirmed by immunofluorescent stain of α-smooth muscle actin (α-SMA) with MMP-9 CD treatment. Furthermore, salvianolic acid B (SalB) inhibited the effects of MMP-9 CD significantly.

In conclusion, our results provide evidence for a direct influence of MMP-9 on cardiac fibroblast migration, collagen and cytokine secretion, which can be attenuated by SalB.

Introduction

Cardiac fibroblasts, which account for 60–70% of the cells in the heart, are critical not only to normal myocardial function, but also in the remodeling that occurs in response to hypertension, myocardial infarction and heart failure (Souders et al. 2009). Cardiac fibroblasts respond to these pathological stimuli by altering their proliferation, migration, modifying extracellular matrix turnover, and modulating secretion of bioactive molecules (Dobaczewski and Frangogiannis 2009). Although these changes serve initially as an important reparative wound healing response, in the longer term they become maladaptive leading course to net accumulation of collagen, cardiac fibrosis and loss of cardiac function (Takeda et al. 2010).

During the cardiac remodeling process, ordinarily quiescent fibroblast transforms into a proliferative and invasive myofibroblast phenotype and initiates structural changes to the cardiac interstitium, which is regulated in part by matrix metalloproteinases (MMPs) (Porter et al. 2004). The MMP family is comprised of more than 25 individual members and is divided into several classes based on in vitro substrate specificity for various extracellular matrix components (Roy et al. 2009). MMP-9 activity is increased during cardiac injury, and targeted deletion of MMP-9 attenuates myocardial remodeling in mice (Romanic et al. 2002). However, there is no evidence about the direct regulation of MMP-9 on fibroblast migration, proliferation, collagen synthesis, cytokine secretion and myofibroblast phenotype transition.

Salviae miltiorrhizae, one of the most important traditional herbal medicines, has been widely used in clinic in China, Japan, and other countries for the treatment of cardiovascular diseases (Li and Wan 2004). As the most abundant and bioactive ingredient, SalB (Fig. 1) has raised a considerable concern and been assigned as the marker component of Salviae miltiorrhizae in the Chinese Pharmacopoeia. Our previous study revealed that the extraction of Salviae miltiorrhizae, which containing 65% SalB, inhibited the activity of MMP-9 instead of MMP-2 at myocardium of rat with myocardial infarction (Jiang et al. 2009a). It is unknown whether SalB itself regulates the effects of MMP-9 on cardiac fibroblast.

Given these facts, the purposes of this study are to investigate the effects of MMP-9 on proliferation, migration, collagen synthesis, cytokine secretion of cardiac fibroblasts, and the roles of SalB during these progresses.

Section snippets

Construction of catalytic domain of MMP-9

The full-length MMP-9 cDNA (MGC-12688) was purchased from American Type Culture Collection. The truncated catalytic domain of MMP-9 (MMP-9 CD) comprising residues 107–216 and 391–444 was constructed and cloned into a pET-15b vector (Novagen Brand), resulting in expression plasmid pET15b-MMP-9 CD (Jiang et al. 2010). The recombinant protein MMP-9 CD was expressed in E. coli. BL21 (DE3) strain using pET15b-MMP-9 CD. The inclusion bodies were dissolved in 50 mM Tris–HCl (pH 7.5), 10 mM CaCl2, 500 mM

MMP-9 CD stimulated the proliferation of cardiac fibroblast and the effects of SalB

Cell proliferation was detected using CCK-8 kit according description of manufacture. As shown in Fig. 1A, MMP-9 CD stimulated the proliferation of fibroblast from 20 nM to 20 μM, and 200 nM was the concentration for weakest stimulation (0.40 ± 0.02 versus 0.34 ± 0.02, p < 0.05). Influence of proliferation should be eliminated as much as possible for migration experiment, so 200 nM MMP-9 CD was chosen for the following experiment. As shown in Fig. 1B, the absorbance for indicated concentration of SalB

Discussion

Cell migration and invasion play a key role in numerous pathological conditions including wound healing, atherosclerosis, cancer, cardiac infarction, and MMPs were suggested to facilitate cell migration and invasion through degrading the extracellular matrix (Kakkar and Lee, 2010, Salo et al., 1994). However, the direct regulation of MMPs on cardiac fibroblast is still poorly elucidated. In the present study, there are three new major findings. First, MMP-9 CD could directly stimulate

Acknowledgments

This work was supported by National Science & Technology Major Project for “Key New Drug Creation and Manufacturing Program” (2009ZX09308-005; 2009ZX09102-122; 2009ZX090304-002; 2009ZX09311-001; 2009ZX09502-020), partly supported by Major Projects of Knowledge Innovation Program of the Chinese Academy of Science (KSCX2-YW-R-166).

References (27)

  • B. Goldberg et al.

    An analysis of collagen secretion by established mouse fibroblast lines

    J. Cell Biol.

    (1964)
  • B. Jiang et al.

    Salvianolic acid B functioned as a competitive inhibitor of matrix metalloproteinase-9 and efficiently prevented cardiac remodeling

    BMC Pharmacol.

    (2010)
  • B. Jiang et al.

    Cardioprotection and matrix metalloproteinase-9 regulation of salvianolic acids on myocardial infarction in rats

    Planta Med.

    (2009)
  • Cited by (48)

    • Tongguan capsule derived-herb ameliorates remodeling at infarcted border zone and reduces ventricular arrhythmias in rats after myocardial infarction

      2019, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Compared with TGC, TGD has a stronger inhibitory effect on VT susceptibility after MI (Supplementary file). Salvianolic acid B [14], salvianolic acid A [15], caffeic acid [39], or astragaloside IV [40], have antifibrotic effects in different organ fibrosis models, including cardiac fibrosis, which explains the antifibrotic effect of TGD [41]. Salvianolic acid B alleviates angiotensin II-induced cardiac fibrosis via suppressing the nuclear transcription factor-kappa B pathway in vitro [42].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text