Krüppel-Like Factor 15 Modulates CXCL1/CXCR2 Signaling-Mediated Inflammatory Response Contributing to Angiotensin II-Induced Cardiac Remodeling

Abstract

Inflammation is involved in cardiac remodeling. In response to pathological stimuli, activated cardiac fibroblasts (CFs) secreting inflammatory cytokines and chemokines play an important role in monocyte/macrophage recruitment. However, the precise mechanism of CF-mediated inflammatory response in hypertension-induced cardiac remodeling remains unclear. In the present study, we investigated the role of transcription factor Krüppel-like factor 15 (KLF15) in this process. We found that KLF15 expression decreased while chemokine CXCL1 and its receptor CXCR2 expression increased in the hearts of angiotensin II (Ang II)-infused mice. Compared to the wild-type mice, KLF15 knockout (KO) mice aggravated Ang II-induced cardiac hypertrophy and fibrosis. Deficiency of KLF15 promoted macrophage accumulation, increase of CXCL1 and CXCR2 expression, and mTOR, ERK1/2, NF-κB-p65 signaling activation in the hearts. Mechanistically, Ang II dose- dependently decreased KLF15 expression and increased CXCL1 secretion from cardiac fibroblasts but not cardiac myoblasts. Loss- or gain-of-function studies have shown that KLF15 negatively regulated CXCL1 expression through its transactivation domain (TAD). Intriguingly, the adenovirus-mediated full length of KLF15-but not KLF15 with TAD deletion overexpression-markedly prevented pathological change in Ang II-infused mice. Notably, the administration of CXCR2 inhibitor SB265610 reversed KLF15 knockout-mediated aggravation of cardiac dysfunction, remodeling, and inflammation induced by Ang II. In conclusion, our study identifies that KLF15 in cardiac fibroblasts negatively regulates CXCL1/CXCR2 axis-mediated inflammatory response and subsequent cardiac remodeling in hypertension.

Keywords: cardiac remodeling; hypertension; inflammation; renin-angiotensin system; transcription factor.

FIGURE 1

Ang II induced decrease of KLF15 expression associating with increase of CXCL1/CXCR2 expression. Mice were infused with saline or Ang II (1,000 ng/kg/min) for the indicated time. (A) Cardiac KLF15 mRNA was detected and analyzed by qPCR. (B) KLF15 protein expression was detected and analyzed by Western Blot. (C) Representative immunofluorescence image of KLF15 in mice heart section (upper) and quantification analysis (lower). (D) CXCL1 mRNA was measured and analyzed by qPCR. (E) CXCR2 protein was detected and analyzed by Western Blot. *P < 0.05 and ***P < 0.001.

FIGURE 2

Deficiency of KLF15 aggravated Ang II-induced cardiac remodeling. Wild-type (WT) mice and KLF15 knockout (KO) mice were infused with saline or Ang II for 14 days. (A) Representative heart size, WGA stain, Masson stain, and α-SMA immunofluorescence image. (B) Statistical analysis of heart weight/body weight ratio. (C) Quantification analysis of myocyte section area. (D) Quantification analysis of fibrotic area measured by Masson stain. (E) Quantification analysis of α-SMA positive area measured by immunofluorescence. (F) Statistical analysis of blood pressure. (G–I) qPCR analysis of mRNA levels of ANP, BNP, Collagen 1a1, and CXCL1. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 3

KLF15 regulated CXCR2-mediated inflammatory cell infiltration and downstream signal. WT and KO mice were infused with saline or Ang II for 14 days. (A) Representative immunohistology and immunofluorescence image of F4/80, CXCR2 positive cell in heart. (B,C) Quantification analysis of F4/80, CXCR2 positive cell infiltration in mice heart. (D,E) Western Blot and quantification analysis of CXCR2/GAPDH. (F–J) Western Blot and quantification analysis of P-mTOR/T-mTOR, P-ERK1/2/T-ERK1/2 and P-p65/T-p65 of mice heart. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 4

KLF15-TAD negatively regulated CXCL1 transcription in CF. CF and H9c2 cells were stimulated by different doses of Ang II for 24 h. (A,B) KLF15 level was measured and quantified by Western Blot analysis. (C,D) The CXCL1 protein in cell supernatant of Ang II treated CF and H9c2 cells was detected by an Elisa assay. (E) CF cells were transfected with con-siRNA and KLF15-siRNA. Successful knockout of KLF15 by KLF15-siRNA was confirmed by Western Blot. (F) CXCL1 mRNA levels of Ang II-treated WT CF and KLF15 knockdown CF were measured by qPCR. (G) Adenovirus-mediated overexpression of KLF15 and deletion of transactivation domain (TAD) of KFL15 were verified by Western Blot. (H) Luciferase assay was used to detect CXCL1 promoter activity in AdCTL, AdKLF15, and AdKLF15-ΔTAD-infected CFs. (I) CXCL1 mRNA levels of AdCTL, AdKLF15, and AdKLF15-ΔTAD infected CFs were measured and analyzed by qPCR. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 5

KLF15 improved Ang II-induced cardiac remodeling through TAD. AdCTL, AdKLF15 and Ad KLF15-ΔTAD-infected mice were infused with saline or Ang II for 14 days. (A) Representative heart size, WGA stain, Masson stain and α-SMA immunofluorescence image. (B) Statistical analysis of heart weight/body weight ratio. (C) Quantification analysis of myocyte section area. (D) Quantification analysis of fibrotic area measured by Masson stain. (E) Quantification analysis of α-SMA positive area measured by immunofluorescence. (F) Statistical analysis of blood pressure. (G–I) qPCR analysis of mRNA levels of ANP, BNP, Collagen 1a1 and CXCL1. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 6

Inhibition of CXCR2 rescued KLF15 KO-aggravated cardiac remodeling. KLF15 KO mice was i.p. injected with SB265610 (2 mg/kg/day) and infused with Ang II for 14 days. WT and KLF15 KO mice only infused with Ang II were used as control. (A) M-mode echocardiography of left ventricular chamber. (B) Measurement of ejection fraction (EF%) and fractional shortening (FS%). (C) Representative heart size, WGA stain, Masson stain and α-SMA immunofluorescence image. (D) Heart weight/body weight ratio. (E) Quantification of myocyte section area. (F) Quantification of fibrotic area revealed by Masson staining. (G) Quantification of α-SMA positive area. (H) Statistical analysis of blood pressure. (I,J) qPCR analysis of mRNA levels of Collagen 1a1, ANP and BNP. (K,L) Western Blot and quantification analysis of CXCR2/GAPDH. (M–P) Western Blot and quantification analysis of P-mTOR/T-mTOR, P-ERK1/2/T-ERK1/2 and P-p65/T-p65 of mice hearts. *P < 0.05, **P < 0.01, and ***P < 0.001. (Q) A working model describing that KLF15 in cardiac fibroblasts negatively regulates CXCL1/CXCR2 axis-mediated inflammatory response and subsequent cardiac remodeling in hypertension.