Cartilage intermediate layer protein 1 (CILP1): A novel mediator of cardiac extracellular matrix remodelling

Figure 1. CILP1 expression in human cardiac tissue. (A) Increased CILP expression in human myocardial infarct tissue. CILP1 protein was detected in human myocardial tissue by Western blot analysis. RA; remote area tissue, MI; myocardial infarct tissue. Note that the tissue samples from RA and MI were obtained from the same human subject. Cartilage was included as a positive control for CILP1. (B) Western blot of endomyocardial biopsies from patients with aortic valve stenosis and control subjects. Lane 1, Control subject spiked with 1 ng recombinant CILP1; Lanes 2–7 Aortic valve stenosis (AS) patients; Lanes 8–10, control subjects. (C) Quantification of the CILP1 signal from the Western blot shown in B. The signal of CILP1 was corrected for the corresponding GAPDH signal. CILP1 protein levels are shown as mean ± SEM, relative to control subjects. (D) Gene expression analysis of cardiac biopsies from human aortic valve stenosis patients (n = 11) and patients undergoing CABG (n = 6). Pearson correlations were calculated between CILP1-expression and ejection fraction (%), and the expression of the following genes: MYH6, TGFB3, COL1A1, COL1A2, COL3A1, TSP2, BGN and MGP. All expression levels…

Figure 2. Cardiac left ventricular expression level of CILP1, CTGF, Col1a1, TGFβ1 and TGFβ3 in sham animals (C, n = 6) and in the infarct tissue (MI) and remote area (RA) of animals at 5 days (n = 4) and 28 days (n = 7) after induction of a myocardial infarction (MI). Data were normalized to the housekeeping gene Cyclophilin-A using the comparative threshold cycle (Ct) method by calculating 2ΔCt. Data are expressed relative to the sham animals as mean ± SEM. **p < 0.01; ***p < 0.001.

Figure 3. CILP1 expression levels in isolated cardiac cells. (A) CILP1 mRNA levels in adult rat cardiomyocytes (CMC) and cardiac fibroblasts (CFB) (n = 8–14 isolations). (B) CILP1 mRNA levels in neonatal rat cardiomyocytes (CMC) and cardiac fibroblasts (CFB) (n = 8–14 isolations). (C) CILP1 and (D) Col1A1 expression during isolation of adult rat cardiac fibroblasts; ARCT: adult rat cardiac tissue (n = 3), ARCC: adult rat cardiac cells (after collagenase, but before adhesion to culture flasks, n = 4), ARCF: adult rat cardiac fibroblasts (immediately after 1 hour attachment to culture flasks, n = 3). (E and F) Effect of serum-starvation and TGFβ1 on ACTA2 and CILP1expression levels in adult rat cardiac ventricular fibroblasts (n = 5 separate experiments). (G) Effect of TGFβ type 1 receptor inhibitor SB431542 for 24 hrs on adult rat cardiac fibroblast CILP1 expression levels in the presence of serum (n = 4). Data were normalized to the housekeeping gene Cyclophilin-A using the comparative threshold cycle (Ct) method by calculating 2ΔCt and multiplied by 1000 (formula 1000 * 2ΔCt), to enhance readability. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01.

Figure 4. Effect of TGFβ1-incubation for 24 h on gene expression levels of CILP1, CTGF, Col1a1 and TGFβ1 in human cardiac atrial fibroblasts isolated from 3 patients. Data were normalized to the housekeeping gene Cyclophilin-A using the comparative threshold cycle (Ct) method by calculating 2ΔCt and multiplied by 1000 (formula 1000 * 2ΔCt), to enhance readability. Data are expressed as mean ± SEM. *p < 0.05.

Figure 5. Inhibitory effect of CILP1 on TGFβ1 signalling. (A) Inhibitory effect of recombinant CILP1 on TGFβ1-induced ACTA2 overexpression in adult rat CFB (treatment 24 hrs, data from 6 separate cell isolations). (B) Inhibitory effect of recombinant CILP1 on TGFβ1-induced CTGF overexpression in adult rat CFB (treatment 24 hrs, data from 6 separate cell isolations). Data from A en B were normalized to the housekeeping gene Cyclophilin-A using the comparative threshold cycle (Ct) method by calculating 2ΔCt and multiplied by 1000 (formula 1000 * 2ΔCt), to enhance readability. (C) Relative αSMA protein level as determined by ELISA in adult rat CFB treated for 48 hrs with TGFβ1, CILP1 or the combination (data are shown relative to control from 6 separate cell isolations). (D) typical examples of αSMA staining in adult rat CFB incubated for 48 hrs with TGFβ1, CILP1 or the combination. (E) Effect of CILP1 on TGFβ-signalling using a TGFβ promoter/reporter assay. HEK293 cells were cotransfected with CILP1 and the TGFβ promoter/reporter construct, containing SMAD-binding elements (CAGA-Luc) and incubated with TGFβ1 for 24 h before analysis of SMAD-binding activity (data are shown relative to…