Inhibition of FABP4 attenuates cardiac fibrosis through inhibition of NLRP3 inflammasome activation

Abstract

Objectives: Cardiac fibrosis is a key biological process of cardiac remodeling and heart failure. Fatty acid-binding protein 4 (FABP4) is a lipid-binding protein that can regulate glucose and lipid homeostasis, and its expression was elevated in heart failure. However, whether FABP4 is involved in cardiac fibrosis remains unknown.

Materials and methods: The cardiac fibrosis model was established in male C57BL/6 mice with subcutaneously infused angiotensin II (Ang-II) (2.8 mg/kg/day) for 4 weeks. DMSO or FABP4 inhibitor BMS309403 (50 mg/kg/day) was intraperitoneally injected for 4 weeks. Ang II-infused mice, FABP4 inhibitor (BMS309403) injected mice, and ventricular tissue were used for morphological studies, and histological and biochemical analyses (FABP4 protein composition and expression).

Results: Ang II infusion increased FABP4 mRNA and protein expression in the mouse ventricular tissue. After treatment with FABP4 inhibitor BMS309403 for 4 weeks, mice showed improved cardiac structure and function as detected by echocardiography. BMS309403 suppressed cardiac and systemic inflammatory response, reduced collagen deposition, and mRNA expression of collagen type I (COL1A1) and collagen type III (COL3A1) in Ang II-infused mice. BMS309403 also reduced the number of α-smooth muscle actin (α-SMA)+cells and decreased the mRNA expression of α-SMA, matrix metalloproteinases-2 (MMP-2), MMP-9, and transforming growth factor-β (TGFβ) in ventricular tissue.

Conclusion: The inhibitory effect of BMS309403 on cardiac fibrosis might be associated with inhibition of NLRP3 inflammasome activation, which Ang II activated. Thus, our data speculated that inhibition of FABP4 could significantly induce cardiac fibrosis.

Keywords: Cardiac fibrosis; Collagen; FABP4; Fibroblast; MMP-2; MMP-9; NLRP3 inflammasome.

Figure 1

FABP4 expression is elevated in cardiac fibrosis mice. Mice were infused with Ang II (2000 ng/kg/min) for 4 weeks. (a) Representative images of FABP4 in the ventricular muscle of Ang-II-induced mice by immunohistochemistry. Magnification: ×200. (b) RT-qPCR detects FABP4 mRNA levels in the ventricular muscle of mice with and without Ang II infusion. (c) Representative protein bands of FABP4 in Ang-II-induced mice model. (d) Quantification of FABP4 protein levels (n=8 in each group). Data are presented as mean±SD. ***P<0.001 vs saline group

Figure 2

FABP4 inhibitor suppresses Ang II-induced inflammatory response of cardiac tissue. Mice were infused with Ang II and intraperitoneally injected with FABP4 inhibitor BMS309403 (50 mg/kg, once a day) for 4 weeks. (a) Representative images of the ventricular tissues stained with hematoxylin-eosin (H&E). The arrow indicates the infiltrated inflammatory cells. Magnification: ×200. Systematic inflammation was evaluated by measurement of the serum inflammatory cytokines using ELISA, including (b) TNF-α and (c) IL-6 (n=8 in each group). Data are presented as mean ± SD

Figure 3

FABP4 inhibitor reduces cardiac fibrosis in Ang II-infused mice. (a) Ventricular tissues were stained with Masson’s trichrome to show the collagen fiber (blue in the interstitium). Magnification: ×200. (b) Quantification of the fibrotic area. RT-qPCR was carried out to determine the mRNA expression of collagen gene, (c) COL1A1 (encode collagen I), and (d) COL3A1 (encode collagen III). Data are presented as mean±SD, N=8

Figure 4

FABP4 inhibitor suppresses expression of genes associated with the TGF-β pathway. (a) Immunohistochemistry was carried out, and representative images of α-SMA are shown. Magnification: ×200. RT-qPCR was carried out to determine the mRNA expression of (b) α-SMA, (c) MMP-2, (d) MMP-9, and (e) TGF-β. All mRNA levels are normalized to GAPDH mRNA. (F) ELISA was carried out to measure the serum TGF-β level of mice. n=8 in each group

Figure 5

FABP4 inhibitor suppresses the NLRP3 inflammasome activation. (a) Representative western blot bands of FABP4 and NLRP3 pathway proteins, including (b) FABP4, (c) NLRP3, (d) ASC, (e) pro-caspase-1, and (f) cleaved caspase-1 (n=8 in each group)