Scholarly article on topic 'Cartilage Intermediate Layer Protein 1 Suppresses TGF-β Signaling in Cardiac Fibroblasts'

Cartilage Intermediate Layer Protein 1 Suppresses TGF-β Signaling in Cardiac Fibroblasts Academic research paper on "Basic medicine"

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Abstract of research paper on Basic medicine, author of scientific article — Kazuhiro Shindo, Masanori Asakura, Kyung-Duk Min, Shin Ito, Hai Ying Fu, et al.

Summary Background Since transforming growth factor (TGF)-β1-induced cardiac fibrosis following myocardial infarction (MI) leads to heart failure and poor clinical prognosis, we aimed to identify a novel and unknown target for cardiac fibrosis related to the TGF-β signaling. Method and result We performed and investigated RNA-Seq using infarcted mouse hearts, culminating in cartilage intermediate layer protein 1 (CILP1). Interestingly, Cilp1 expression was increased along with TGF-β1 expression in infarcted hearts, and was also upregulated after TGF-β1 stimulation in cardiac fibroblasts in vitro. Histological analysis revealed that Cilp1 was localized at the fibrotic regions of infarcted hearts. Full length CILP1 (F-CILP1) was cleaved into both N-terminal CILP1 (N-CILP1) and C-terminal CILP1 at the furin cleavage site, and both F-CILP1 and N-CILP1 were extracellularly secreted. We further found that CILP1 bound to TGF-β1 via thrombospondin type 1 domain, and suppressed both smad3 phosphorylation and fibroblasts differentiation to myofibroblasts induced by TGF-β1. Conclusion We identified CILP1 as a potential regulator of cardiac fibrosis by inhibiting TGF-β signaling, and these results suggest the promise of CILP1 as a novel therapeutic target for preventing cardiac fibrosis and heart failure in MI patients.

Academic research paper on topic "Cartilage Intermediate Layer Protein 1 Suppresses TGF-β Signaling in Cardiac Fibroblasts"

International Journal of Gerontology xxx (2017) 1—8

Contents lists available at ScienceDirect

International Journal of Gerontology

journal homepage: www.ijge-online.com

Original Article

Cartilage Intermediate Layer Protein 1 Suppresses TGF-ß Signaling in Cardiac Fibroblasts*

Kazuhiro Shindo 1, Masanori Asakura 2 *, Kyung-Duk Min 3, Shin Ito 3, Hai Ying Fu 3, Satoru Yamazaki1, Ayako Takahashi 3, Miki Imazu 1, Hiroki Fukuda 1, Yuri Nakajima 1, Hiroshi Asanuma 4, Tetsuo Minamino 5, Seiji Takashima 6, Naoto Minamino 7, Naoki Mochizuki 1, Masafumi Kitakaze 3

1 Department of Cell Biology, National Cerebral and Cardiovascular Center, Osaka, 2 Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, 3 Department of Clinical Research and Development, National Cerebral and Cardiovascular Center, Osaka, 4 Department of Internal Medicine, Meiji University of Integrative Medicine, Kyoto, 5 Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, Kagawa, 6 Department of Medical Biochemistry, Osaka University Graduate School of Medicine, 7 Omics Research Center, National Cerebral and Cardiovascular Center, Osaka, Japan

ARTICLE INFO

SUMMARY

Article history: Received 13 January 2017 Accepted 16 January 2017 Available online xxx

Keywords: cardiac fibroblast, cardiac fibrosis,

cartilage intermediate layer protein 1, myocardial infarction, transforming growth factor-b

Background: Since transforming growth factor (TGF)-b1-induced cardiac fibrosis following myocardial infarction (MI) leads to heart failure and poor clinical prognosis, we aimed to identify a novel and unknown target for cardiac fibrosis related to the TGF-b signaling.

Method and result: We performed and investigated RNA-Seq using infarcted mouse hearts, culminating in cartilage intermediate layer protein 1 (CILP1). Interestingly, Cilpl expression was increased along with TGF-P1 expression in infarcted hearts, and was also upregulated after TGF-P1 stimulation in cardiac fibroblasts in vitro. Histological analysis revealed that Cilp1 was localized at the fibrotic regions of infarcted hearts. Full length CILP1 (F-CILP1) was cleaved into both N-terminal CILP1 (N-CILP1) and C-terminal CILP1 at the furin cleavage site, and both F-CILP1 and N-CILP1 were extracellularly secreted. We further found that CILP1 bound to TGF-P1 via thrombospondin type 1 domain, and suppressed both smad3 phosphorylation and fibroblasts differentiation to myofibroblasts induced by TGF-P1. Conclusion: We identified CILP1 as a potential regulator of cardiac fibrosis by inhibiting TGF-b signaling, and these results suggest the promise of CILP1 as a novel therapeutic target for preventing cardiac fibrosis and heart failure in MI patients.

Copyright © 2017, Taiwan Society of Geriatric Emergency & Critical Care Medicine. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

1. Introduction

Although fibrosis is one of the most essential causes of cardiac remodeling, its precise mechanism remains unclear. In cases of myocardial infarction (MI), cardiac fibrosis causes left ventricular (LV) remodeling, resulting in cardiac systolic and diastolic dysfunction and thus heart failure1. Importantly, LV remodeling is an independent prognostic risk factor for fatal arrhythmia in MI patients2. The renin—angiotensin—aldosterone and sympathetic

* Conflict of interest: All contributing authors declare that they have no conflicts of interest.

* Correspondence to: Masanori Asakura, MD, PhD, Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan. Fax: +81 798 45 6551.

E-mail address: ma-asakura@hyo-med.ac.jp (M. Asakura).

nervous systems, oxidative stress, and inflammatory cytokines are involved in LV remodeling3, which is currently treated with angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, and b-blockers. Transforming growth factor-b (TGF-b) signaling has recently been focused on for developing new treatments for suppressing cardiac fibrosis. Indeed, TGF-b expression levels increase immediately after MI onset4 and causes increased collagen production as it directly acts on cardiac fibroblasts, inducing their differentiation to myofibroblasts. TGF-b also regulates the extracellular matrix by inhibiting matrix destruction via protease inhibitors in the infarcted area1. Thus, TGF-b signaling is an important factor controlling cardiac fibrosis, and new approaches targeting TGF-b signaling are anticipated to decrease cardiac remodeling associated with cardiac fibrosis.

http://dx.doi.org/10.1016/j.ijge.2017.01.002

1873-9598/Copyright © 2017, Taiwan Society of Geriatric Emergency & Critical Care Medicine. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Fig. 1. RNA-Seq analysis of the infarcted area in the MI group. A. The top 15 genes that exhibited large changes in expression in infarcted areas in the MI group, compared with those in the sham group (FDR; P < 0.01). B. The search for cardiac fibrosis-related candidate genes was performed. First of all, the genes reportedly related to TGF-b were identified,

To respond to this urgent requirement, we investigated the changes in gene expressions of the infarcted murine hearts and identified cartilage intermediate layer protein 1 (CILP1) as a new fibrosis-inhibiting factor that suppresses TGF-b signaling by binding to TGF-31. CILP1, mainly expressed in articular cartilage and intervertebral discs, is involved in maintaining intervertebral disc shapes5,6, however the fibrosis-related function of CILP1 remains to be reported in the heart. Therefore, we investigated the role of CILP1 in cardiac fibrosis.

2. Materials and methods

2.1. Antibodies and reagents

TGF-P1 (T7039), phenylephrine (PE; P6126), angiotensin II (AngII; A9525), endothelin 1 (ET1; E7764), hydrogen peroxide (h2O2; 13-1910-5), and anti-FLAG M2 monoclonal antibody (A8592) were purchased from Sigma-Aldrich (St. Louis, MO). Insulin-like growth factor (IGF-1; 291-G1) was purchased from R&D System (Minneapolis, MN). Antibodies against TGF-b1 (sc146) and anti-mouse IgG (sc2005) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Smad3 (p-Smad3; 9520S), Smad2/3 (8685S), and anti-rabbit IgG (7074S) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against glycer-aldehyde3-phosphate dehydrogenase (Gapdh; HAB374) was purchased from EMD Millipore (Billerica, MA). Cilp1 polyclonal antibody was raised in rabbits against a synthetic peptide (RQTMLAQSVRRVQPVKRTPKTLAKPADSQE) corresponding to pre-proCILP122-51, after conjugating with keyhole limpet hemocyanin via its C-terminal cysteine. Antibodies for immunofluorescence staining of 4', 6-diamidino-2-phenylindole (DAPI) and alpha-smooth muscle actin (a-SMA)-Cy3 (D1306) were purchased from Invitrogen (Carlsbad, CA) and Sigma-Aldrich, respectively.

2.2. MI surgery

Male C57BL/6J mice (8-week-old; weight, 22-24 g; SLC Japan, Shizuoka, Japan) were anesthetized by intraperitoneally injecting 0.3 mg/kg medetomidine hydrochloride, 4 mg/kg midazolam, and 5 mg/kg butorphanol tartrate. MI (n = 5) was induced by ligating the left anterior descending artery7 and compared with the sham group (n = 5). Myocardial samples were obtained on first, 4th, 7th, and 14th day after the onset of MI and corresponding time in the sham group. At the end of the experiment, the hearts were immediately excised, snap frozen, and stored at -80°C for RNA or protein purification. Sham mice were identically prepared without undergoing MI protocols. Animals used for histological analysis underwent 7-day ischemia protocols (four animals per group).

All procedures complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (the 8th Edition, NRC 2011) and were approved by the National Cerebral and Cardiovascular Center Committee for Laboratory Animal Use.

2.3. RNA-Seq analysis

The libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit, according to the manufacturer's instructions (Illumina, Inc., San Diego, CA). Single-end sequencing (75 bp) was performed using Hiseq 2500 (Illumina). FASTQ data files were

analyzed using CLC Genomic Workbench software (version 9; CLC bio, Arhus, Denmark). Approximately 20 million raw reads were obtained for each sample. A quality check was performed, and mouse genome mapping was conducted. Expression analysis was performed using the edgeR package8.

2.4. Real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qRT-PCR) was performed as previously described9. In brief, mouse ventricles and cultured neonatal rat cardiac fibroblasts and cardiomyocytes were performed for total RNA isolation using TRIzol reagent (Invi-trogen). TaqMan probes used to quantify Cilpl, Tgf-@1, a-SMA, collagen type I alpha 1 chain (Collagen lal), collagen type I alpha 2 chain (Collagen 1a2), collagen type III alpha 1 chain (Collagen 3a1), and Gapdh. qRT-PCR was performed using the StepOne Real-Time PCR System (Applied Biosystems). The copy number of each gene was normalized to Gapdh by the comparative Ct method10.

Other methods are described in Supplementary information.

2.5. Statistical analysis

Data are expressed as the mean ± SEM. Student's t-test was used for comparing the two groups. All data, except RNA-Seq data, were analyzed with JMP 12.2 software (SAS Institute, Cary, NC), and P < 0.05 was considered significant.

3. Results

3.1. RNA-Seq of mouse hearts in the MI group and investigation of Cilpl expression

To investigate new TGF-b-related genes in MI, myocardial samples from the infarct area were analyzed on day 7 after MI. Expression analysis was performed for each sample: We selected top 15 genes with large expression changes and sought the genes related to fibrosis as candidate genes (Fig. 1A). Ten genes were related to TGF-b. And among these 10 genes, 9 genes were already reported in the heart. Therefore, only one gene, Cilp1, was left (Fig. 1B).

Next, MI hearts were used to confirm Cilp1 expression using qRT-PCR. In the MI group, samples were obtained from infarcted, peri-infarcted, and noninfarcted areas. In the sham group, samples were obtained from the LV anterior wall. Cilp1 expression increased from day 4 in the MI group, peaking on day 7 to levels 29 times higher than those in the sham group, before subsequently decreasing. Compared with the sham group, Cilp1 expression levels increased in MI hearts, including infarcted, peri infarcted and noninfarcted areas (Fig. 1C). Similar to Cilp1, Tgf-@1, Collagen 1a1, Collagen 1a2, and Collagen 3a1 expression levels increased in MI hearts, peaking on day 7 and subsequently decreasing (Fig. 1D-G, respectively). a-SMA expression levels were the highest in the infarcted areas and continued increasing until day 14, instead of a peak on day 7 (Fig. 1H).

3.2. Cilp1 expression observed in infarcted areas and cultured cardiac fibroblasts

Immunostaining was performed on tissues from the MI border region, and Cilp1 expression sites were confirmed. Strong Cilp1

culminating in 10 genes. After exclusion of the genes that appeared in heart-related reports, one gene was left: Cilp1 was identified as a novel cardiac fibrosis-related gene related to TGF-P1. C. On days 1,4,7, and 14 after MI, Cilp1 expression was confirmed in infarcted areas (Inf), peri-infarcted areas (Peri), and noninfarcted areas (Non) in the MI group, and in the LV anterior wall in the sham group using qRT-PCR (MI: n = 5, sham: n = 5). *P < 0.05 compared with the sham group.

expression was observed in the infarcted areas in the MI group along with almost no Cilp1 expression in the sham group (Fig. 2A). Western blotting analysis showed that Cilp1 expressed only in the samples from the infarcted, but not the peri-infarcted or no infarcted area (Fig. 2B).

Next, because TGF-ß16 and IGF-111 induce CILP1 in cartilage cells, AngII induces fibrosis12, phenylephrine induces cardiac hypertrophy13, ET1 expression level increases during MI14, and H2O2 oxidative stress increases during MI15, these reagents were used to confirm whether Cilpl was expressed in cardiac fibroblasts or car-diomyocytes. Using qRT-PCR, Cilp1 expression was observed when TGF-ß1 was administered to neonatal rat cardiac fibroblasts. However, no Cilp1 expression was detected using the other reagents or in cardiomyocytes (Fig. 2C), indicating that Cilp1 expression level increases in infarcted areas and that its expression is induced by TGF-ß1 in cardiac fibroblasts.

3.3. In cardiac fibroblasts, CILP1 binds to TGF-fbl through thrombospondin type 1 (TSP1) and suppresses Smad3 phosphorylation

In cartilage cells, because CILP1 inhibits Smad2 phosphorylation by binding to TGF-316, we decided to confirm whether TGF-b signaling was suppressed in cardiac fibroblasts. The three FLAG-tagged vectors, expressing full-length (F-), N-terminal (N-) and C-terminal (C-) CILP1 (Fig. 3A) were transfected into HEK293T cells, and the expression was confirmed in the supernatant and cell lysate. After the overexpression of F-CILP1, F-CILP1 and cleaved N-CILP1 and C-CILP1 were found in the cell lysate, whereas the secretion of F-CILP1 and N-CILP1, but not C-CILP1, were found in the supernatant. N-CILP1-FLAG was expressed at the same level as N-CILP1 cleaved from the F-CILP1-FLAG. However, C-CILP1 expression was detected only in the cell lysate, but not the supernatant. These

Fig. 2. Cilp1 expression increased in the infarcted area in the MI group. A. Hematoxylin—eosin staining, picrosirius red staining, and Cilp1 immunostaining in the peri-infarcted areas on day 7 after MI. The dotted line indicates the border between the infarcted and noninfarcted areas (scale bar, 50 mm). B. On day 7, Cilp1 expression in the infarcted areas (Inf), peri-infarcted areas (Peri), and noninfarcted areas (Non) was confirmed by Western blotting. In the sham group, sites in the heart equivalent to each site used in the MI group were used. C. TGF-P1 (10 ng/mL), IGF-1 (100 nmol/L), AngII (100 nmol/L), PE (100 mmol/L), ET1 (100 nmol/L), and H2O2 (20 mmol/L) were administered to neonatal rat cardiac fibroblasts or cardiomyocytes, and Cilp1 expression was evaluated using qRT-PCR. Triplicate experiments were performed. *P < 0.05 compared with the sham group.

Fig. 3. CILP1 inhibited Smad3 phosphorylation in cardiac fibroblasts. A. The Flag tags were added to the C terminus to create full-length CILP1 (F-CILP1-FLAG), N-terminal domain CILP1 (N-CILP1-FLAG), and C-terminal domain CILP1 (C-CILP1-FLAG), and the TSP1 domain was removed from N-CILP1 to create N-CILP1-TSP1(- )-FLAG constructs. B. F-CILP1-FLAG, N-CILP1-FLAG, and C-CILP1-FLAG were transfected into HEK293T cells, and Western blotting was used to evaluate the expression of F-, N-, and C-CILP in cells and supernatant after 24 h. C. After incubating TGF-31 (10 ng/mL) and F-CILP1-FLAG (100 mg/mL) at room temperature for 1 h, the constructs were administered to neonatal rat cardiac fibroblasts, and the cells were harvested after 20 min. D. After incubating TGF-31 (10 ng/mL) with N-CILP1-FLAG (100 mg/mL) or C-CILP1-FLAG (100 mg/mL) at room temperature for 1 h, the constructs were administered to neonatal rat cardiac fibroblasts, and the cells were harvested after 20 min. E. TGF-31 (10 ng/mL) was incubated with N-CILP1-FLAG or N-CILP1-TSP1(-)-FLAG at room temperature, and immunoprecipitation was performed with anti-TGF-31 antibodies. F. After incubating TGF-31 (10 ng/mL) with N-CILP1-FLAG (100 mg/mL) or N-CILP1-TSP1(-)-FLAG (100 mg/mL) at room temperature for 1 h, the constructs were administered to neonatal rat cardiac fibroblasts and cells were harvested after 20 min.

results indicated that F-CILP1 was cleaved into N-CILP1 and C-CILP1 in the cells and that F-CILP1 and N-CILP1 were secreted from cells to supernatant (Fig. 3B).

As TGF-3 promotes Smad3 phosphorylation16, we investigated the effects of F-CILP1-FLAG on TGF-3 signaling. Purified F-CILP1-FLAG was mixed with TGF-b1 and administered to the neonatal rat cardiac fibroblasts. F-CILP1 inhibited Smad3 phosphorylation induced by TGF-31 in a dose-dependent manner (Fig. 3C).

To confirm whether N-CILP1 or C-CILP1 inhibited TGF-b signaling, purified N-CILP1-FLAG and C-CILP1-FLAG were mixed with TGF-31, respectively, and administered to the neonatal rat cardiac fibroblasts. N-CILP1-FLAG inhibited Smad3 phosphorylation, whereas C-CILP1-FLAG did not (Fig. 3D).

Since CILP1 has a TSP1 domain, which has been shown to bind TGF-316, we constructed a vector expressing N-CILP1 without TSP1 domain (N-CILP1-TSP1(-)) (Fig. 3A) to determine whether CILP1 would bind to TGF-31 and inhibit its signaling pathway. N-CILP1-FLAG and N-CILP1 -TSP1(- )-FLAG were mixed with TGF-31, respectively, and immunoprecipitation was performed using anti-TGF-31

antibodies. Binding to TGF-31 occurred with N-CILP1 -FLAG in a dose-dependent manner, but no binding occurred with N-CILP1-TSP1(-)-FLAG (Fig. 3E). Moreover, N-CILP1, but not N-CILP1-TSP1(-), inhibited Smad3 phosphorylation induced by TGF-31 (Fig. 3F). These results demonstrated that in cardiac fibroblasts, CILP1 bound to TGF-31 via the TSP1 domain to inhibit Smad3 phosphorylation.

3.4. CILP1 inhibits TGF-@1-induced a-SMA in cardiac fibroblasts

Cardiac fibroblasts are stimulated by TGF-31 to differentiate to myofibroblasts and produce a-SMA17. Therefore, we evaluated whether CILP1 would inhibit the differentiation induced by TGF-31. TGF-31 was mixed with N-CILP1-FLAG, C-CILP1-FLAG, and N-CILP1-TSP1(-)-FLAG, respectively, and then was administered to the cardiac fibroblasts of neonatal rats. TGF-31 enhanced a-SMA expression, which was suppressed by N-CILP1, but not C-CILP1 or N-CILP1-TSP1(-) (Fig. 4A). Consistently, a-SMA expression induced by TGF-31, as confirmed by qRT-PCR, was also inhibited by N-CILP1, but not C-CILP1 or N-CILP1-TSP1 (-) (Fig. 4B). These results

Fig. 4. In cardiac fibroblasts, CILP1 inhibited a-SMA induction through TGF-01. After incubating TGF-P1 (10 ng/mL) with N-CILP1-FLAG (N-CILP1) (100 mg/mL), C-CILP1-FLAG (C-CILP1) (100 mg/mL), or N-CILP1-TSP1(-)-FLAG [N-CILP1-TSP1(-)] (100 mg/mL) at room temperature for 1 hour, the constructs were administered to neonatal rat cardiac fibroblasts. Twenty-four hours later, a-SMA expression was evaluated by immunofluorescence staining (A) and qRT-PCR (B). Scale bar = 50 mm in A. Experiments were performed in triplicates in B. *P < 0.05 compared with the vehicle-treated group. #P < 0.05 compared with the TGF-b1-treated group.

demonstrate that CILP1 inhibits TGF-b1 via the TSP1 domain, suppressing the differentiation of fibroblasts to myofibroblasts.

4. Discussion

The present study revealed that 1) Cilp1 expression is increased in post-MI fibrotic foci, 2) Cilp1 is mainly expressed in fibroblasts, 3)

CILP1 binds to TGF-ß1 through the TSP1 domain, and 4) CILP1 inhibits TGF-ß signaling in cardiac fibroblasts, suppressing their differentiation to myofibroblasts.

CILP1 has been previously reported to be expressed in articular cartilage and intervertebral discs, contributing to the maintenance of disc shapes5,6. In cartilage cells, either TGF-b1 or IGF-1 induces CILP1 expression11,18. TGF-ß1 induces CILP expression mainly

through Smad3 phosphorylation18. In nucleus pulposus cells, CILP1 directly binds to TGF-31 and suppresses its signals6. For patients with lumbar disc diseases, C allele (coding for Thr 395) of 1184T/C increased binding and inhibition of TGF-31. Therefore, its allele depresses collagen production and increases prevalence for lumbar disc diseases6,19. We similarly indicated that CILP1 suppressed TGF-3 signaling through TSP1 domain in cardiac fibroblasts. We also found that CILP1 expression can be increased by TGF-31 stimulation and controlled by negative feedback mechanisms. In addition, in the infarcted hearts, this action may be exaggerated when car-diomyocytes become necrotic and cardiac fibroblasts grow in infarcted area of MI. Importantly we found that CILP1 is secreted by cardiac fibroblasts, not cardiomyocytes. It makes sense that CILP1 has a negative feedback function and is secreted by cardiac fibro-blasts in infarcted areas for TGF-3 signaling suppression. This negative feedback system is similar in cartilage cells. However, CILP1 is constantly expressed in cartilage cells and maintains ho-meostasis of cartilage. In the heart, CILP1 is induced only during the pathologic period. Anti-TGF-3 therapy for MI might develop adverse effect depending on the timing of the therapy20,21. In other words, when the therapy for cardiac fibrosis is conducted as targeting TGF-3 signaling, timing of CILP1 expression may possibly be an indicator for therapeutic medicine, and CILP1 can also be expected as a new marker for anti-TGF-3 therapy.

In the heart, TGF-3 signaling induces the differentiation of fi-broblasts in post-MI fibrotic foci to myofibroblasts and promotes collagen production17,22. In addition, TGF-3 regulates the extracellular matrix synthesis by inhibiting matrix destruction and inducing protease inhibitors in infarcted areas1. It also functions as a key regulator of fibrosis in heart diseases, especially in heart failure23. Therefore, new treatment agents targeting TGF-3 signaling promise to improve cardiac remodeling associated with cardiac fibrosis. Therapy as targeting TGF-3 signaling for cardiac fibrosis suppression has already been developing, but it is not practically available yet. For instance, TGF-3 neutralizing antibody was used for suppressing cardiac remodeling with certain time window for murine MI, but it could contrarily increase mortality and worsen LV remodeling20,21. One plausible explanation is that TGF-3 has another function to inhibit inflammation and neutro-philic migration to endothelium or even activate monocyte during the early stage of MI. It also promotes cardiac fibrosis as inducing collagen production from cardiac fibroblasts during the chronic phase. The diverse, multifunctional, and pleiotropic effects of TGF-3 on cells involved in infarct healing1. Thus, it is important to identify an endogenous factor controlling TGF-3 function in tissue- or time-specific manners. We could successfully identify CILP1 which is endogenous protein controlling TGF-3 using the heart samples of MI model. Because CILP1 is induced in fibroblasts (Fig. 2) and extracellularly secreted (Fig. 3B), CILP1 may possibly become a new cardiac fibrosis marker. Moreover CILP, as an endogenous protein, may suppress cardiac fibrosis with minimum adverse effects, which provided a new approach for MI treatment by inhibiting TGF-31.

The other novelty of the current study is that CILP1 holds TSP1 domain. Domain including WSXW motif would be specifically bound active form of TGF-3124. We demonstrated that N-CILP1 with removed TSP1 was not bound with TGF-31 (Fig. 3E). Collagen tissue growth factor (CTGF), another TGF-3 signal-related protein with TSP1 domain, is also increased after MI25. Furthermore, it is reported that CTGF regulates angiogenesis by binding to vascular endothelial growth factor (VEGF) through TSP1 domain in endo-thelial cells26. Although we did not examine the binding of CILP1 to VEGF in the present study, CILP1 may possibly control angiogenesis by affecting endothelial cell other than inhibitory action on cardiac fibrosis. Thus, further investigations are needed to clarify the role of CILP1 in MI therapy.

Cilp1 expression increased in the heart after MI. Furthermore, CILP bound to TGF-ß via TSP1 domain and appeared to suppress cardiac fibrosis by inhibiting TGF-ß signaling, which is strongly involved in MI fibrosis. Our findings provide new opportunities for research into novel treatment targets for cardiac fibrosis.

Acknowledgments

The authors would like to thank Ms. Akiko Ogai for excellent technical assistance and Dr. Osamu Tsukamoto for technical advice in CILP1 purification.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ijge.2011.01.002.

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