2021 Volume 69 Issue 2 Pages 178-184
C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) plays a crucial part in cardiovascular diseases especially the coronary artery disease. Nonetheless, it is unrevealed that whether the CTRP12 participates in the progress of cardiac fibrosis. In this study, we investigated whether CTRP12 regulates pathological myocardial fibrosis. We isolated neonatal rat cardiac fibroblasts were cultured with recombination CTRP12 followed by stimulating with Isoproterenol (ISO, 100 µM) for 24 h. Then the adenovirus were used to achieve the CTRP12-overexpressed fibroblasts. In vivo, the C57/B6 mice were subjected to recombinant human CTRP12 (0.2 µg/g/d) for 2 weeks after injected with Isoproterenol (ISO, 10 mg/kg/d for 3 d then 5 mg/kg/d for 11 d, subcutaneously (s.c.), 2 weeks) and mice were also subjected to adenovirus with P38 overexpressing system to explore the mechanism. As a result, CTRP12 significantly inhibit the transformation of cardiac fibroblasts to myofibroblasts and the transcription of cardiac fibrosis-related proteins induced by ISO in vitro. The administration of CTRP12 can effectively reduce the cardiac fibrosis and enhance the cardiac function in mice hearts. The treatment with CTRP12 did not change the expression level of phosphorylated (p)-smad2, smad4, p-extracellular regulated protein kinases 1/2 and c-Jun N-terminal kinase 1/2, but it suppressed the activation of p38. Cardiac overexpression of p38 could abolish this kind of cardioprotective effects by CTRP12. In summary, the CTRP12 protect against the ISO induced cardiac fibrosis via suppressing the p38 signal pathway.
Cardiovascular diseases (CVD) are the primary cause of death worldwide, and even worse the death ratio caused by CVD are higher and continues to grow in many of the developing countries.1) Cardiac fibrosis, as a common pathological process of most CVD, contributes to mechanical dysfunction and structural remodeling of the heart driven by multiple pathogenic factors.2) Initially, fibrosis is a compensatory regulation for replacing the dead cardiomyocytes. But taking into account the long-term affect, within fibrotic hearts, the cardiac biochemical, electrical and mechanical homeostasis can be gradually disrupted by the abnormal accumulation of extracellular matrix proteins in the myocardial interstitial.3) Based on the increasing evidence which unveiled the partly underlying mechanisms of fibrosis, we know that the process is promoted together by various kinds of cells (myofibroblasts; cardiomyocytes; vascular cells; some inflammatory cells), mixture pathogenic factors (proinflammatory cytokines; growth factors, reactive oxygen species, Endothelin-1 etc.) as well as neuroregulatory mechanism (renin/angiotensin/aldosterone system).4) Unfortunately, there are lack of efficient strategies which can postpone or reverse the cardiac fibrotic alterations.
C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) or Adipolin is an adipokine candidate which is mainly expressed in white adipose tissue. A lot of researches about CTRP12 all focus on its potential capacity on curing type2 diabetes mellitus. More than most adipokines [tumour necrosis factorα (TNFα), Monocyte chemoattractant protein-1 (MCP-1) and interleukin6 (IL6)], the concentration of CTRP12 is down-regulated in both fat tissue of obese animals and plasma of obesity animals.5) Moreover, increasing CTRP12 expression can release the inhibited insulin sensitivity, shown anti-inflammation benefit and recover the metabolism homeostasis by ameliorating glucose metabolism and increasing glucose clearance in obese animal models.6,7)
The complex relationship between obesity and cardiovascular disease is not fully understood. But as we known, a variety of bioactive molecules (adipokines/adipocytokines) secreted by adipose tissue can effect on circulation and heart. Nowadays, limited researches look into the functions of CTRP12 in the cardiovascular diseases. Hayato8) and his colleagues reported that CTRP12-knockout can attenuate the process of pathological vascular remodelling via anti-inflammation function and inhibiting the proliferation of vascular smooth muscle cell in the mice with injured femoral artery. Reza’s9) group found out that serum levels of CTRP12 are independently associated with coronary artery disease (CAD) and several CAD risk factors. But nothing is provided about whether there is some link between CTRP12 and pathogenic mechanisms of cardiac fibrosis, such as inflammation and classic fibrosis relevant pathways.
Cell counting assay (CCK8) was purchased from Beyotime (Shanghai, China). Isoproterenol was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). α-Smooth muscle actin (SMA) and PCNA antibodies were purchased from Abcam, U.S.A. Antibodies including phosphorylated (P)-smad2, tatal (T)-smad2, smad4, P-extracellular regulated protein kinases (ERK), P-c-Jun N-terminal kinase (JNK), P-p38, and T-ERK, T-JNK, T-p38, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology Inc. (Danvers, MA, U.S.A.).
AnimalsC57BL6J male mice ages 8–10 weeks (weighted 23.5–27.5 g) were purcahsed from Chinese Academy of Medical Sciences & Peking Union Medical College. The animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996) and were approved by the Animal Care and Use Committee of our hospital. Mice were subjected to isoproterenol (ISO) injection (3 d with 10 mg/kg/d, 11 d with 5 mg/kg/d for consecutively 14 d, subcutaneously). In the final injection of ISO, mice were then subjected to recombinant mouse CTRP12 infusion (0.2 µg/g/d) with an Alzet osmotic minipump (Durect, Cupertino, CA, U.S.A.) for 2 weeks. Then mice were sacrificed and hearts were removed.
Echocardiography MeasurementsEchocardiography were detected after the final infusion of CTRP12. A Mylab30CV (ESAOTE) ultrasound System was used. M-mode tracings and pulse doppler were recorded by a 15 MHz probe. Left ventricular (LV) ejection fraction (LVEF), LV fractional shortening (LVFS), E/A ratio were calculated.
Preparation of Recombinant Mouse CTRP12 ProteinFull-length mouse CTRP12 cDNA was transfected into HEK293F cells by a pCDNA3.1 vector with the Targefect-293F reagent. HEK293F cells were cultured. We use a His Ni-NTA resin to collect and purify CTRP12.
Mice Overexpression of p38The adenovirus vector that overexpress p38 (Ad-p38) and the negative control (Ad-NC) was constructed by Vigene Biosciences, INC. (Shanghai, China). One week before ISO injection, Mice were subjected to retroorbital venous plexus injection of Ad-p38 (each with 60 µL, 5.0–6.5 × 1013 GC/mL) as previous described.10)
Histological AnalysisHearts were removed and fixed in 10% formalin, then embedded in paraffin and cutted into 25 µm section. Picrosirius Red staining was performed to detect LV collagen deposition. We used a digital image analysis system (Image-Pro Plus, version 6.0) to analysis PSR-stained sections.
Fibroblasts Isolation and CultureAdult (4–6 weeks) C57BL6J mice were scarrifaced and hearts were removed. We used a 0.125% trypsin and collagenase to digestion (5 times, each 8 min). Digestions were collected in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium containing 15% fetal bovine serum (FBS). Cells were then centrifugated and re-suspend in DMEM/F12 medium containing 10% FBS and filtered in a 40 µm sieve. Then cells were seeded in a 10 cm dish. After 90 min adherence, we removed non-fibroblasts that not in adherent. Cell were then stimulated with ISO (100 µM, diluted in phosphate buffered saline (PBS), the control cells were treated with same volume of PBS) for 24 h and then treated with CTRP12 (0.5, 1, 2, 4, 8 µg/mL, diluted in PBS containing 0.1% bovine serum albumin (BSA)) for 24 h. The cells in vehicle group were treated with same volume of PBS containing 0.1% BSA. Cell were also transfected with CTRP12 adenovirus to overexpress CTRP12 (MOI = 100). To inhibit P38, cells were treated with P38 inhibitor (SB209063, 10 mM) for 8 h. CCK8 assay was used to detect cell proliferation.
RT-PCR and Western BlotWe used TRIzol reagent o isolate total mRNA in fibroblasts. We used the SmartSpec Plus Spectrophotometer (Bio-Rad, Hercules, CA, U.S.A.) to detect mRNA purity with OD260/OD280 ratios. A total of 2 µg of mRNA was reverse transcribed into cDNA with cDNA Synthesis Kit (Roche Diagnostics, Switzerland). We used a LightCycler 480 SYBR Green I Master kit (Roche Diagnostics) to amplifications. We used GAPDH as reference. The primers used are listed below:
For Western blot, we used radioimmunoprecipitation (RIPA) lysis buffer to lysis cells. Total protein concentration was detected by a bicinchoninic acid (BCA) method and then loaded to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). When proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, U.S.A), they were incubated with primary antibodies in 4 °C overnight. Primary antibodies included: CTRP12 (Biocaompany, 1 : 500 dilution), phosphorylated (P-) and total AKT, smad2, ERK1/2, JNK1/2, P38 (all from Cell signaling Technology, 1 : 1000 dilution), smad4, adipoR1, adipoR2, and GAPDH (all purchased from Abcam and used at 1 : 1000 dilution). Blots were developed with enhanced chemiluminescence (ECL) reagents (Bio-Rad) and captured by a ChemiDoc MP Imaging System (Bio-Rad). We used GAPDH as reference.
Immunoflurorence StainingCell were fixed with 4% paraformaldehyde for 5 min and then permeabilized with 0.1% Triton™ X-100. PCNA and antibody (abcam, 1 : 100 dilution) were used to incubate cells for 1h in 37 °C, then cells were cultured in a Alexa FluorH 568 goat anti-rabbit Immunoglobulin IgG (Invitrogen Life Technologies, Carlsbad, CA, U.S.A.). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain nuclears.
Data AnalysisAll data were expressed as mean ± standard deviation (S.D.). SPSS 23.3 were used to analysis data. When comparing data with two groups, a Student t test was used. When comparing data among four groups, a one-way ANOVA followed by Tukey’s post hoc test was used. We defined significantly different with p value less than 0.05.
We first used a recombination mouse CTRP12 to treat adult mice fibroblasts after cells were stimulated with ISO. As shown in Fig. 1A, cells undergoing robust growth when stimulated with ISO for 24 h. CTRP12 (0.5, 1, 2, 4, 8 µL/mL) could inhibited this growth. We then explore fibroblast’s activation via α-SMA staining, since activated fibroblast expressing more α-SMA, which make it more contractile. As we observed in Fig. 1A, cells in ISO group expressed more α-SMA, while cells treated with CTRP12 (8 µL/mL) were expressed less α-SMA. After activation fibroblast synthetises more collagen protein, we then detect the transcription level of those collagen proteins. As observed in Fig. 1C, increased collagen I, colagen III and CTGF were found in ISO stimulated cells while CTRP12 remarkablely reduced the mRNA level of those protein. Fibroblasts rapidly proliferation is another feature of fibrosis. We stained fibroblasts with PCNA a proliferation marker. ISO induced a more PCNA expression in fibroblasts, while CTRP12 dropped these up-regulating in reponse to ISO stimuli (Fig. 1D). Taken togeter, these data indicates that CTRP12 could inhibit ISO stimulated fibroblasts activation, proliferation and collagen synthesis in vitro.
A. Cell counting kit-8 detecting cell proliferation in adult mice fibroblasts stimulated with ISO (100 µM) and treated with CTRP12 (0.5, 1, 2, 4, 8 µg/mL) (* p < 0.05 vs. CON; # p < 0.05 vs. ISO). B. α-SMA inmmunoflorense staining in cells stimulated with ISO (100 µM) and treated with CTRP12 (8 µg/mL). C. Transcription level of collagen proteins in cells stimulated with ISO (10 nM) and treated with CTRP12 (8 µg/mL). D. PCNA inmmunoflorense staining in cells stimulated with ISO (100 µM) and treated with CTRP12 (8 µg/mL). ** p < 0.01. All the in vitro experiments were repeated independently for 3 times.
We then explore whether CTRP12 overexpression in fibroblasts would exert the same effects as Mouse recombination CTRP12. We transfected cells with Ad-CTRP12 after ISO stimulation to overexpress CTRP12 (Fig. 2A). Then cells proliferation was detected by CKK8 assay. As expected, ISO induced fibroblasts proliferation, activation, collagen synthesis were all blurred by CTRP12 overexpression (Figs. 2A–E). These data suggestes that CTRP12 may exert anti-cardiac fibrosis effect in disease model.
A. CTRP12 expression level after cells were transfected with Ad-CTRP12. B. Cell counting kit-8 detecting cell proliferation in adult mice fibroblasts stimulated with ISO and transfected with Ad-CTRP12. C. α-SMA inmmunoflorense staining in cells. D. Transcription level of collagen proteins in cells. E. PCNA inmmunoflorense staining in cells. ** p < 0.01. All the in vitro experiments were repeated independently for 3 times.
Mice were subjected to ISO injection to establish cardiac fibrosis model and then administrated to CTRP12 infusion for 2 weeks. In ISO group, we observed a significant LV collagen deposition as shown by PSR staining. While the LV collagen volume was much less in CTRP12-ISO group than in vehicle-ISO group (Figs. 2A, B). We also observed same trend of mRNA levels of collagen I, collagenIII and CTGF in ISO injected mice hearts (increase in vehicle-ISO group, dropped in CTRP12-ISO group, Fig. 3C). Cardiac function was evaluated by echocardiography at the end of CTRP12 infusion. As shown in Fig. 3D, LVEF, LVFS, and E/A ratio were dropped in vehicle-ISO group, but increased in CTRP12-ISO group. The heart rate was increased in vehicle-ISO group and we did not observed a difference between vehicle-ISO group and CTRP12-ISO group.
Mice were subjected to ISO injection for 14 d and then subjected to CTRP12 infusion for another 2 weeks. A and B. PSR staining and quantitative results (n = 6). C. Transcription level of collagen proteins in hearts. D. Echocardiographic measurements in mice (n = 8). ** p < 0.01.
We performed Western blot to screen the possible signaling that may affect by CTRP12. Smad signaling is the typical pro-fibrosis pathway. But we did not observe a change in AKT, smad2 and smad4 between vehicle-ISO group and CTRP12-ISO group (Figs. 4A, B). We then explore other fibrosis pathway. Mitogen-activated protein kinases (MAPKs) is another typical non-smad signlaing that promotes fibrosis. We found that ISO induced increase in ERK, JNK and p38 phosphorylation level, but CTRP12 merely decreased p38 phosphorylation level (Figs. 4C, D). These suggestes that CTRP12 may affects cardiac fibrosis via regulating p38 activation.
A and B. P-AKT, T-AKT, P-smad2, T-smad2, smad4 protein levels in in fibroblasts stimulated with ISO (100 µM) and treated with CTRP12 (8 µg/mL). C and D. P-ERK, T-ERK, P-JNK, T-JNK, P-p38, and P-p38 protein levels in in fibroblasts stimulated with ISO (100 µM) and treated with CTRP12 (8 µg/mL). ** p < 0.01. All the in vitro experiments were repeated independently for 3 times.
Previous study reported that the receptor of CTRP12 is adiponectin receptor (Adipo R).11) Thus we assess whether fibroblasts express CTRP12 affect Adipo R. As shown in Fig. 5A, both Adipo R1 and Adipo R2 expressed in fibroblasts. But, CTRP12 treatment did not affect the expression of Adipo R1 and Adipo R2. To comfirm whether P38 signaling could affect ISO induced fibroblasts activation. P38 inhibitor (SB209063, 10 mM) was used. As a result, P38 inhibitor suppressed ISO induced fibroblasts proliferation, α-SMA expression, and collagen synthesis (Figs. 5C–F). These suggest that P38 exerts a pro-fibrosis effect in cardiac fibroblasts.
A and B. The protein lecel of Adipo R1 and Adipo R2 in cells stimulated with ISO (100 µM) and treated with CTRP12 (8 µg/mL). C–F. Cells were treated timulated with ISO (100 µM) and treated with P38 inhibitor (SB209063, 10 mM). A. Cell counting kit-8 detecting cell proliferation. B. α-SMA inmmunoflorense staining. C. Transcription level of collagen proteins. D. PCNA inmmunoflorense staining. * p < 0.05. All the in vitro experiments were repeated independently for 3 times.
Wherther p38 overexpression would counteract CTRP12’s effects? We then injected Ad-P38 in mice to overexpression p38. Both P-p38and T-P38 were elevated in Ad-P38 mice hearts (Fig. 6A). And mice then subjected to 14 d of ISO injection and 2 weeks of CTRP12 infusion. We found that in mice with Ad-P38 injection, the LV collagen volume was more serious than CTRP12 infusion group. And collagen proteins transcription level was more than CTRP12 infusion group. Cardiac funciton in Ad-P38 injection group was deteriorated than CTRP12 infusion group (Figs. 6B–D). These indicates that p38 overexpression obscures CTRP12’s anti-fibrosis effects.
Mice were subjected to Ad-p38 injection 1 week before ISO injection, then subjected to ISO injection for 14 d and then CTRP12 infusion for another 2 weeks. A. P-p38 and T-p38 level in mice heart after 5 weeks of Ad-p38 injection (n = 6). B. PSR staining and quantitative results (n = 6). C. Transcription level of collagen proteins in hearts. D. Echocardiographic measurements in mice (n = 8). * p < 0.05; ** p < 0.01.
CTRP12 an insulin-sensitizing adipokine was associated with many diseases such as obese, Type 2 diabetes mellitus,12) coronary artery disease9) and pathological vascular remodeling.8) However, the effect of CTRP12 on cardiac fibrosis was unclear. In this study, we first elucidate the protective role of CTRP12 on cardiac fibrosis induced by ISO insult both in vivo and vitro. We reveal that both mouse recombination CTRP12 and overexpress CTRP12 by adenovirus could suppress ISO induced fibroblasts activation, collagen synthesis and proliferation. Mice with CTRP12 infusion could ameliorate ISO induced cardiac fibrosis and improve cardiac function. These anti-fibrosis effects of CTRP12 may lead it to a new therapeutic methods for many cardiovascular disease.
More than 800000 people worldwide die each year from fibrotic diseases, including cardiac fibrosis.13) During the pathology of cardiovascular disease, especially myocardial infarction and other sort of cardiac injury, fibrosis leads to scar formation.13) On the one hand this process guarantees the structural integrity of the heart; on the other hand, it reduces the compliance of the heart, and reduces electrical conduction, resulting in abnormal cardiac contraction and diastolic function.14) Secondly, long-term high-volume loading can also lead to myocardial fibrosis, which eventually leads to cardiac pumping disorders.15) During the development of cardiac fibrosis, the activation of resident fibroblasts in heart tissue contributes to the majority of these pathological changes. Under insult, fibroblasts activates to myofibroblasts, which highly express α-SMA with enhanced cell contractility. These myofibroblasts synthesize a large amount of collagen fibers, proliferate in large quantities, and secrete a variety of profibrotic factors, which in turn induces fibroblast proliferation and collagen synthesis.16) As an adipokine, most of the studies have focused on the metabolic regulation function of CTRP12.12,17,18) However, recently, Hiramatsu-Ito and colleagues reported that CTRP12 suppressed vascular cell inflammation and proliferation in injured arteries.8) These give us a clue that CTRP12 may affect on fibroblasts proliferation and activation. In this study, we found that CTRP12 could inhibit ISO induced fibroblasts activation, collagen synthesis, as well as proliferation. We also used a ISO stimulated cardiac fibrosis mice model to confirm the functional role of CTRP12 on cardiac fibrosis in vivo. Consistently, we have got the positive result that CTRP12 just inhibited the established cardiac fibrosis and cardiac dysfunction.
When screening the underlying mechanism, we first focus on the most common fibrotic signaling smad. Once activated by transforming growth factor (TGF) β, smad2, smad3 were phosphorylated and then binds to smad4 to form a heterotrimer.14) These complex then move the the nuclear and binds to the relative DNA promoter region to enhance the fibrotic gene transcription, such as collagen, α-SMA, and CTGF.19) But, in our experiments, we did not observe a change in smad2 and smad4 by CTRP12, despite these proteins were all elevated after ISO stimulation. We then turned to the second common pathway MAPKs. MAPKs is a smad independent pathway that leads to fibrosis. The activation of TGFβ type II receptor leads to the downstream target TGF beta-Activated Kinase 1 (TAK1) and GTPase Ras, which subsequently activates JNK, p38, and ERK pathway.20,21) The pro-fibrosis efffects of these MAPKs protein was confirmed by gene knockout experiments, in which knockout of those MAPKs could block fibroblasts activation and proliferation in many disease models.14,22,23) In Ogawa’s study, they found that CTRP12 regulated vascular remodeling via TGF-βRII/Smad2-dependent pathway.8) But in our study, we did not found an effect of CTRP12 on smads but observed a change in p38 protein (not JNK and ERK). We also confirmed these effect by using p38 overexpressing adenovirus in vivo. As p38 overexpression counteracted the anti-fibrosis role of CTRP12. Our data confirm that CTRP12 exert anti-fibtosis role via p38 regulation. Previous study reported that the receptor of CTRP12 is Adipo R.11) whether fibroblasts express CTRP12 affect Adipo R. In our study, both Adipo R1 and Adipo R2 expressed in fibroblasts. But, CTRP12 overexpression did not affect the expression of Adipo R1 and Adipo R2. We suspect more CTRP12 expression may increase the banding of itself with Adipo R, suppress p38 signaling.
In summary, our study demonstrated for the first time that CTRP12 could ameliorate ISO induced cardiac fibrosis and improve cardiac function. CTRP12 exert these effects by directly function on fibroblasts via p38 regulation. And further study about the application of CTRP12 on fibrosis in cardiovascular disease are urgetly needed in order to develop a new therapeutic mehtod.
The authors declare no conflict of interest.
