Cardiac Fibrosis Is Associated With Decreased Circulating Levels of Full-Length CILP in Heart Failure

Figures

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  Figure 1. Identification of Potential Biomarkers for Cardiac Fibrosis (A) Volcano plot depicting highly differentially expressed genes in cardiac fibroblasts after transverse aortic constriction (TAC). (B) Heat map of profibrotic genes that are up-regulated in cardiac fibroblasts isolated from sham and TAC mouse hearts. Select genes (Ltbp2, Comp, and Cilp) are boxed. (C) Reverse transcription–quantitative polymerase chain reaction confirmation of up-regulation of latent transforming growth factor-β binding protein 2 (Ltbp2), cartilage oligomeric matrix protein (Comp), and cartilage intermediate layer protein 1 (Cilp) in cardiac fibroblasts 7 days post-TAC (7D TAC) (n = 3). *p < 0.050 (Student’s t-test). Acta2 = α-smooth muscle actin; Angptl4 = angiopoietin-like 4; Col5a2 (1a1, 1a2, 2a1,3a1, 4a2, 5a1) = collagen type V (I, II, III, IV) α-2 (α-1); Ctgf = connective tissue growth factor; Ddah1 = dimethylarginine dimethylaminohydrolase 1; Edn1 = endothelin 1; Fn1 = fibronectin 1; Lox = lysol oxidase; Mfap5 = microfibrillar associated protein 5; Mmp14 = matrix metallopeptidase 14; Postn = periostin; Ptn = pleitrophin; RPKM = reads per kilobases mapped; Serpinh1 = serine (or cysteine) peptidase…

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  Figure 2. Ltbp2, Comp, and Cilp Are Up-Regulated in Cultured Cardiac Fibroblasts After TGFβ Treatment (A) Schematic of cardiac fibroblast culture and transforming growth factor(TGF)-β treatment. (B) Reverse transcription–quantitative polymerase chain reaction of cardiac fibroblasts treated with TGFβ normalized to untreated control samples (indicated by dotted line at y = 1) (n = 4). (C) Immunocytochemistry for Ltbp2, Comp, and Cilp show significant increase in expression after TGFβ treatment (n = 4). We used 4′,6-diamindino-2-phenylindole (DAPI) (blue) stain for nuclei. Bar = 100 μm. *p < 0.050 (Student’s t-test). Abbreviations as in Figure 1.

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  Figure 3. Ltbp2, Comp, and Cilp Localize to the Fibrotic Myocardium (A) Mice that had undergone TAC surgery exhibited myocardial fibrosis as shown by Masson trichrome staining. (B) Immunofluorescence staining shows colocalization of Ltbp2, Comp, and Cilp (red) with fibroblast marker discoidin domain receptor tyrosine kinase 2 (Ddr2) (green, left) and activated fibroblast marker α-smooth muscle actin (SMA) (green, right). Red channel images are shown separately for clarity. DAPI (blue) stain was used for nuclei. Bars = 50 μm. Abbreviations as in Figures 1 and 2.

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  Figure 4. LTBP2, COMP, and CILP are Up-Regulated in Human Cardiac Fibroblasts in Response to TGF-β (A) Ribonucleic acid sequencing data from a public database (GSE46224) show that expression of LTBP2, COMP, and CILP are up-regulated in heart biopsies from patients with ischemic heart failure (HF) compared with biopsies from healthy patients. (B) Schematic of culture and TGF-β treatment of human cardiac fibroblasts. (C) Reverse transcription–quantitative polymerase chain reaction of human cardiac fibroblasts treated with TGF-β normalized to untreated control samples (indicated by dotted line at y = 1) (n = 3). (D) Immunocytochemistry shows increased expression of LTBP2, COMP, and CILP in cultured human cardiac fibroblasts after TGF-β treatment. DAPI (blue) stain was used for nuclei (n = 4). Bar = 100 μm. *p < 0.050 (Student’s t-test). Abbreviations as in Figures 1 and 2.

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  Figure 5. LTBP2, COMP, and CILP Are Up-Regulated in the Myocardium of Patients With HF (A) Masson trichrome staining show extensive fibrosis in heart sections of patients with heart failure. Insets are higher magnification images of boxed area. (B) LTBP2, COMP, and CILP (red) expression is significantly increased in hearts undergoing heart failure. DAPI (blue) stain was used for nuclei. Bars = 50 μm. Abbreviations as in Figures 1, 2, and 4.

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  Figure 6. Full-Length CILP Is Downregulated in the Serum of Patients With HF (A) Enzyme-linked immunosorbent assay demonstrates nonsignificant changes in levels of COMP in the serum of patients with heart failure (n = 22) compared with healthy individuals (n = 23). (B) Patients with heart failure (n = 22) had a significant decrease in circulating CILP levels compared with the levels in healthy individuals (n = 23). (C) Representative Western blotting shows decreased levels of full length CILP (∼133 kDa) in serum of patients with heart failure. (D) Quantified data are shown for healthy individuals (n = 5) and patients with heart failure (n = 5). *p < 0.050 (Student’s t-test). AU = arbitrary units; other abbreviations as in Figures 1 and 4.