Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling.

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

The matricellular protein CCN5 prevents adverse atrial structural and electrical remodelling.

Atrial structural remodelling including atrial hypertrophy and fibrosis is a key mediator of atrial fibrillation (AF). We previously demonstrated that the matricellular protein CCN5 elicits anti-fibrotic and anti-hypertrophic effects in left ventricles under pressure overload. We here determined the utility of CCN5 in ameliorating adverse atrial remodelling and arrhythmias in a murine model of angiotensin II (AngII) infusion. Advanced atrial structural remodelling was induced by AngII infusion in control mice and mice overexpressing CCN5 either through transgenesis (CCN5 Tg) or AAV9-mediated gene transfer (AAV9-CCN5). The mRNA levels of pro-fibrotic and pro-inflammatory genes were markedly up-regulated by AngII infusion, which was significantly normalized by CCN5 overexpression. In vitro studies in isolated atrial fibroblasts demonstrated a marked reduction in AngII-induced fibroblast trans-differentiation in CCN5-treated atria. Moreover, while AngII increased the expression of phosphorylated CaMKII and ryanodine receptor 2 levels in HL-1 cells, these molecular features of AF were prevented by CCN5. Electrophysiological studies in ex vivo perfused hearts revealed a blunted susceptibility of the AAV9-CCN5-treated hearts to rapid atrial pacing-induced arrhythmias and concomitant reversal in AngII-induced atrial action potential prolongation. These data demonstrate the utility of a gene transfer approach targeting CCN5 for reversal of adverse atrial structural and electrophysiological remodelling.

Effect of glucose, soya oil and glutamine on protein expression and mammalian target of rapamycin complex 1 pathway of jejunal crypt enterocytes in weaned piglets.

The present study was conducted to evaluate the effects of glucose, soya oil or glutamine on jejunal morphology, protein metabolism and protein expression of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway in jejunal villus or crypt compartment of piglets. Forty-two 21 d-weaned piglets were randomly allotted to one of the three isoenergetic diets formulated with glucose, soya oil or glutamine for 28 d. On day 14 or 28, the proteins in crypt enterocytes were analysed with isobaric tags for relative and absolute quantification and proteins involved in mTORC1 signalling pathway in villus or crypt compartment cells were determined by Western blotting. Our results showed no significant differences (P > 0·05) in jejunal morphology among the three treatments on day 14 or 28. The differentially expressed proteins mainly took part in a few network pathways, including antimicrobial or inflammatory response, cell death and survival, digestive system development and function and carbohydrate metabolism. On day 14 or 28, there were higher protein expression of eukaryotic initiation factor-4E binding protein-1 in jejunal crypt compartment of piglets supplemented with glucose or glutamine compared with soya oil. On day 28, higher protein expression of phosphor-mTOR in crypt compartment was observed in piglets supplemented with glucose compared with the soya oil. In conclusion, the isoenergetic glucose, soya oil or glutamine did not affect the jejunal morphology of piglets; however, they had different effects on the protein metabolism in crypt compartment. Compared with soya oil, glucose or glutamine may be better energy supplies for enterocytes in jejunal crypt compartment.

Effects of corn particle size on nutrient utilization in pigs evaluated under optimal and heat stress conditions.

The effects of corn particle size on nutrient digestibility and energy utilization in pigs were determined under optimal (experiment 1, 25 ± 1 °C) or heat stress (experiment 2, 37 ± 1 °C) conditions. In Exp. 1 and 2, five experimental diets were tested using a 5 × 5 Latin square design involving five barrows (Landrace × Yorkshire × Duroc, average initial body weight of 30 ± 1 kg and 45.0 ± 1.8 kg, respectively, in individual metabolic cages). Dietary treatments were as follows: 200-, 300-, 400-, 600-, 800-µm corn particle sizes obtained by mesh screens. Under optimal thermal conditions, digestibility of dry matter (DM) and crude fiber (CF) from 200-µm diet was higher (P < 0.05) compared to that from the 300-µm and 400-µm diets. The digestibility of crude protein (CP) and ether extract (EE) was the highest (P < 0.05) at the 200-µm particle size. The apparent total tract digestibility of energy was significantly higher (P < 0.05) on the 200-µm diet. Under heat stress, digestibility of CF when corn was ground to 600 µm was higher (P < 0.05) compared to 300 and 400 µm. Digestibility of NDF and ADF was the highest (P < 0.05) at 600-µm corn particle size. In conclusion, grinding corn to 200-µm corn particles had a positive effect on DM, CP, EE, and CF under optimal thermal condition, while the 600-µm corn particle size had positive effects on digestibility of CF, NDF, and ADF than 200-µm corn particle size under heat stress.

Integral imaging system using an adaptive lens array.

We produced an adaptive lens array composed of multiple flat lens arrays arranged in a curved shape with an adjustable radius of curvature, in order to overcome the hardware problem of the conventional flat or curved lens array-based systems. The manufactured adaptive lens array is applied to an integral imaging system. The gap mismatch that occurs when using a curved lens array is resolved by computing the exact display mapping position of element images through each lens. The results of the experiment demonstrate that the adaptive lens array-based integral imaging system successfully generated elemental images according to the curvature transformation of the adaptive lens array, and they were reconstructed as 3D images.

A learning curve in aortic dissection surgery with the use of cumulative sum analysis.

This study demonstrates the risk adjusted cumulative sum analysis of an individual surgical learning curve for acute type A aortic dissection surgery. Thirty consecutive patients were operated by a single surgeon for acute type A aortic dissection from April 2001 to March 2008. Operative variables, mortality, and major morbidities were analyzed. The learning curve was calculated by cumulative sum analysis. The anticipated 30-day operative mortality rate was 20.2 +/- 12.7% (range, 3.3-56.7%) and the expected 30-day mortality and morbidity rate was 47.0 +/- 13.3% (range, 21.7-70.6%) according to the Japan SCORE calculator. The observed operative and in-hospital mortality rate was 6.67% (two patients), and the observed major postoperative morbidity rate was 10.0% (three patients). Risk-adjusted cumulative sum analysis revealed that no excess deaths occurred beginning at the seventh case and thereafter. The surgeries for acute type A aortic dissection could be performed at the professionally permissive level from the beginning. Risk-adjusted cumulative sum analysis was a useful tool to monitor the performance of the surgical procedure.

Risk-adjusted mortality rate cumulative sum analysis based on the Japan SCORE represents a learning curve in mitral valve reparative surgery.

PURPOSE: We calculated the cumulative sum analysis of an individual surgical learning curve for mitral valve reconstructive surgery to monitor quality control. METHODS: Between April 2003 and March 2009, 25 consecutive patients underwent mitral valve reconstructive surgery for mitral regurgitation (MR). All operations were performed by a single surgeon. We analyzed the operative variables, mortality, and major morbidities and calculated the learning curve, using descriptive statistics and cumulative sum analysis based on the Japan SCORE calculator. RESULTS: In this series, the Japan SCORE calculator predicted a 30-day operative mortality rate of 3.07 ± 3.48 % and a 30-day operative mortality and morbidity rate of 14.9 ± 9.58 %s. In reality, there was no operative or in-hospital death and the major postoperative morbidity rate was 6.0 % (two patients). The risk-adjusted cumulative sum analysis revealed that the learning curve for mortality and morbidity shifted downward below the lower confident interval for all the patients, on completion of mitral valve repair. Coefficient analysis revealed a significant negative correlation between the aortic cross-clamp time and the cardiopulmonary time and case load. There was no correlation between the operation time and the case load. CONCLUSIONS: Routine reconstructive mitral valve surgery for MR can be performed at the professionally permissive level from the beginning, even in low-volume case loads.

The matricellular protein CCN5 prevents anti-VEGF drug-induced epithelial-mesenchymal transition of retinal pigment epithelium.

Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly worldwide. Anti-vascular endothelial growth factor (VEGF) drugs have been widely used to treat the neovascular type of AMD (nAMD). However, VEGF acts not only as a pro-angiogenic factor but also as an anti-apoptotic factor in the eyes. In this study, we found that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells in vitro, accompanied by the induction of CCN2, a potent pro-fibrotic factor. Similarly, intravitreal injection of Afl into mouse eyes resulted in EMT in the retinal pigmented epithelium (RPE). Co-treatment with CCN5, an anti-fibrotic factor that down-regulates CCN2 expression, significantly attenuated the adverse effects of the anti-VEGF drugs both in vitro and in vivo. Inhibition of the VEGF signaling pathway with antagonists of VEGF receptors, SU5416 and ZM323881, induced EMT and up-regulated CCN2 in ARPE-19 cells. Additionally, knock-down of CCN2 with siRNA abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells. Collectively, these results suggest that anti-VEGF drugs induce EMT in RPE through the induction of CCN2 and that co-treatment with CCN5 attenuates the adverse effects of anti-VEGF drugs in mouse eyes.

Urinary Metabolomic Differentiation of Infants Fed on Human Breastmilk and Formulated Milk.

Human breastmilk is an invaluable nutritional and pharmacological resource with a highly diverse metabolite profile, which can directly affect the metabolism of infants. Application of metabolomics can discriminate the complex relationship between such nutrients and infant health. As the most common biological fluid in metabolomic study, infant urinary metabolomics may provide the physiological impacts of different nutritional resources, namely human breastmilk and formulated milk. In this study, we aimed to identify possible differences in the urine metabolome of 30 infants (1-14 days after birth) fed with breast milk (n = 15) or formulated milk (n = 15). From metabolomic analysis with gas chromatography-mass spectrometry, 163 metabolites from single mass spectrometry (GC-MS), and 383 metabolites from tandem mass spectrometry (GC-MS/MS) were confirmed in urinary samples. Various multivariate statistical analysis were performed to discriminate the differences originating from physiological/nutritional variables, including human breastmilk/formulate milk feeding, sex, and duration of feeding. Both unsupervised and supervised discriminant analyses indicated that feeding resources (human breastmilk/formulated milk) gave marginal but significant differences in urinary metabolomes, while other factors (sex, duration of feeding) did not show notable discrimination between groups. According to the biomarker analyses, several organic acid and amino acids showed statistically significant differences between different feeding resources, such as 2-hydroxyhippurate.

Advantage of a more central incision onto left atrium by using ultrasonic scalpel.

Exposure of the mitral valve is of critical importance in mitral valve surgery. The mitral valve is located near the interatrial groove. As we accumulated experience and learned from embryology, we found that a more central left atriotomy was advantageous, and was made possible by interatrial groove dissection using an ultrasonic scalpel. This crucial finding prompted us to report the technique.

Reappraisal of importance of the left internal mammary artery to the left anterior descending artery in improving mid-term outcome in patients with severe left ventricular dysfunction.

We sought to investigate the effects of the left internal mammary artery anastomosed to the left anterior descending artery for improving mid-term outcome in patients with severe left ventricular dysfunction. Twenty consecutive coronary artery bypass grafts performed by a single surgeon for less than 35% ejection fraction patients were reviewed retrospectively from April 2000 to November 2008. There was one perioperative death (5.0% mortality). The mean survival was 55 months with an actual 5-year survival rate of 75%. Echocardiography showed the ejection fraction improved to 10.7 +/- 0.32% (p < 0.01). The strongest correlation was observed between both the flow and the pulsatile index of the left internal mammary artery measured intraoperatively by transit time flow meter and the postoperative ejection fraction improvement (R2=0.737 and 0.639, respectively). We reappraised the mid-term beneficial effects of the internal mammary artery anastomosed to the left anterior descending artery in patients with severe left ventricular function.

Dynamic modeling of pesticide residue in proso millet under multiple application situations.

Proso millet (Panicum miliaceum L.) is a cereal crop with potential resistance to drought and heat stress, making it a promising alternative crop for regions with hot and dry climates. Because of its importance, it is crucial to investigate pesticide residues in proso millet and assess their potential risks to the environment and human health to protect it from insects or pathogens. This study aimed to develop a model for predicting pesticide residues in proso millet using dynamiCROP. The field trials consisted of four plots, with each plot containing three replicates of 10 m2. The applications of pesticides were conducted two or three times for each pesticide. The residual concentrations of the pesticides in the millet grains were quantitatively analyzed using gas and liquid chromatography-tandem mass spectrometry. The dynamiCROP simulation model, which calculates the residual kinetics of pesticides in plant-environment systems, was employed for predicting pesticide residues in proso millet. Crop-specific, environment-specific, and pesticide-specific parameters were utilized to optimize the model. Half-lives of pesticides in grain of proso millet, which were needed to input for dynamiCROP, were estimated using a modified first-order equation. Proso millet-specific parameters were obtained from previous studies. The accuracy of the dynamiCROP model was assessed using statistical criteria, including the coefficient of correlation (R), coefficient of determination (R2), mean absolute error (MAE), relative root mean square error (RRMSE), and root mean square logarithmic error (RMSLE). The model was then validated using additional field trial data, which showed that it could accurately predict pesticide residues in proso millet grain under different environmental conditions. The results demonstrated the accuracy of the model in predicting pesticide residues in proso millet after multiple applications.

Metabolomic Responses of Lettuce (Lactuca sativa) to Allelopathic Benzoquinones from Iris sanguinea Seeds.

Weed management is important in modern crop protection. Chemical weed control using synthetic herbicides, however, suffers from resistance and ecotoxicity. Metabolomic investigation of allelopathy (or allelochemicals) may provide novel alternatives to synthetic herbicides. This study aimed to investigate the detailed metabolomic responses of plants to allelochemicals in Iris seed extracts. The seed extracts of Iris sanguinea showed the strongest growth inhibitory activity against alfalfa, barnyard grass, lettuce, and mustard. 3-Hydroxyirisquinone (3-[10(Z)-heptadecenyl]-2-hydroxy-5-methoxy-1,4-benzoquinone) was isolated as a major allelochemical from I. sanguinea seeds through bioassay-guided fractionation. The compound inhibited the growth of shoots and roots by browning root tips. Discriminant analysis identified 33 differentially regulated lettuce metabolites after treatment with 3-hydroxyirisquinone (3HIQ). Metabolic pathway analysis revealed that several metabolic pathways, including aromatic amino acid biosynthesis and respiratory pathways, were affected by the compounds. Differential responses of membrane lipids (accumulation of unsaturated fatty acids) and extensive formation of reactive oxygen species were observed in root tissues following treatment with 3HIQ. Overall, alkylbenzoquinone from I. sanguinea induced extensive metabolic modulation, oxidative stress, and growth inhibition. The metabolomic responses to allelochemicals may provide fundamental information for the development of allelochemical-based herbicides.

Dissipation Kinetics and Risk Assessment of Diniconazole, Dinotefuran, Metconazole, and Tebuconazole in Raphanus sativus L.

This study investigated the degradation characteristics and conducted a risk assessment of four pesticides (Diniconazole, Dinotefuran, Metconazole, and Tebuconazole) in the leaves and roots of radish. Radish was cultivated in two greenhouse fields, and samples were collected at 0, 1, 2, 3, 5, 7, and 10 days after pesticide application. Sample analysis was performed using LC-MS/MS, and the recovery rates ranged from 70.1% to 118.6%. The biological half-life of Diniconazole was found to be 6.2 days (leaf and root), Dinotefuran was 5.3 days (leaf) and 4.6 days (root), Metconazole was 9.3 days (leaf) and 3.2 days (root), and Tebuconazole was 8.0 days (leaf) and 5.1 days (root). After comparing the maximum residue limits (MRL) of each pesticide in Korea with the residues during the pre-harvest interval (PHI), Diniconazole showed a Hazard quotient (HQ) exceeding 1, indicating potential risks for true consumers. Furthermore, Tebuconazole showed an HQ of 0.3 or higher, indicating a significant level of risk.

Metabolomic approach of azole fungicides in radish (Raphanus sativus): Perspective of functional metabolites.

Azole fungicides is one of the major fungicides in agricultural field. In this study, toxic effects of diniconazole (DIN), metconazole (MET), and tebuconazole (TEB) to radish leaves and roots were investigated using targeted metabolomics with gas chromatography-mass spectrometry (GC-MS/MS). Especially, the changes of functional chemicals, including phytosterols and glucosinolates evaluated. Radish leaves and roots were harvested after 7 days and 14 days from last exposure. In multivariate analysis, the experimental groups showed clear separation in PCA and PLS-DA score plots. Phytosterols and glucosinolates were significantly changed by azole fungicide. Six metabolic pathways which are affected by fungicides were selected and showed similar patterns regardless of the type of azole fungicide used. As a result, azole fungicide induces the defense mechanisms of plants and affects both primary and secondary metabolism.

Quantitative Profiling of Carotenoids, Tocopherols, Phytosterols, and Fatty Acids in the Flower Petals of Ten Marigold (Tagetes spp. L.) Cultivars.

Marigold (Tagetes spp.) flower petals are the most vital sources of carotenoids, especially lutein esters, for the production of natural lutein to use for food, feed, and pharmaceutical industries. Several marigold cultivars are cultivated globally; however, their lutein ester composition and contents have not been widely investigated. Considering this, this study aimed to identify and quantify prominent carotenoid esters from the flower petals of ten marigold cultivars by liquid chromatography (LC)-diode-array detection (DAD)-mass spectrometry (MS). In addition, tocopherols, phytosterols, and fatty acids were analyzed by gas chromatography (GC)-flame ionization detection (FID) and GC-MS. Furthermore, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging abilities of lipophilic extracts were determined. The total carotenoid contents varied significantly (p < 0. 05, Tukey HSD) among cultivars, ranging from 25.62 (cv. Alaska)-2723.11 µg/g fresh weight (cv. Superboy Orange). Among the five major lutein-diesters, (all-E)-lutein-3-O-myristate-3'-O-palmitate and lutein dipalmitate were predominant. Among the studied cultivars, α-tocopherol was recorded, ranging from 167.91 (cv. Superboy Yellow) to 338.50 µg/g FW (cv. Taishan Orange). Among phytosterols, ß-sitosterol was the most prevalent phytosterol, ranging between 127.08 (cv. Superboy Yellow) and 191.99 µg/g FW (cv. Taishan Yellow). Palmitic acid (C16:0; 33.36-47.43%) was the most dominant among the fatty acids. In this study, the highest contents of lutein were recorded from cv. Superboy Orange; however, due to the substantially higher flower petal yield, the cv. Durango Red can produce the highest lutein yield of 94.45 kg/ha. These observations suggest that cv. Durango Red and cv. Superboy Orange are the ideal candidates for lutein fortification in foods and also for commercial lutein extraction.

Effects of different stocking density and various phytogenic feed additives dosage levels on growing-finishing pigs.

This study was to investigate the effects of different phytogenic feed additives (PFA) dosage levels in growing- finishing pigs stressed by high stocking density. A total of 72 mix sexed 12 weeks growing pigs ([Landrace × Yorkshire] × Duroc) with initial body weight (BW) of 49.28 ± 4.58 kg were used for 8 weeks. There were 3 replicate pens in each treatment group, with 3 pigs per pen. The dietary treatment groups consisted of basal diets in animal welfare density (negative control [NC]), basal diet in high stocking density (positive control [PC]), PC + 0.04% essential oil (ES1), PC + 0.08% essential oil (ES2), PC + 0.10% bitter citrus extract & essential oil (CES1), PC + 0.20% bitter citrus extract & essential oil (CES2), PC + 0.05% grape pomace extract (GP1), PC + 0.10% grape pomace extract (GP2). The reduction of space allowance decreased (p < 0.05) average daily gain, feed efficiency, and digestibility of dry matter, crude protein, and gross energy. Also, the fecal score of PC groups increased (p < 0.05) compared with other groups. Basic behaviors (feed intake, standing, lying) were inactive (p < 0.05) and singularity behavior (biting) was increased (p < 0.10) under high stocking density. There was no difference in blood profile. However, the supplementation of PFA alleviated the negative effects such as reduced growth performance, nutrient digestibility, and some increasing stress indicators in th blood (cortisol) and animal behavior (biting). In conclusion, the negative effect of high stocking density was most effectively mitigated by the normal dosage of the mixture of bitter citrus extract and essential oil additive (CES1).

A selective array activation method for the generation of a focused source considering listening position.

A focused source can provide an auditory illusion of a virtual source placed between the loudspeaker array and the listener. When a focused source is generated by time-reversed acoustic focusing solution, its use as a virtual source is limited due to artifacts caused by convergent waves traveling towards the focusing point. This paper proposes an array activation method to reduce the artifacts for a selected listening point inside an array of arbitrary shape. Results show that energy of convergent waves can be reduced up to 60 dB for a large region including the selected listening point.

Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration due to dystrophin gene mutations. Patients with DMD initially experience muscle weakness in their limbs during adolescence. With age, patients develop fatal respiratory and cardiac dysfunctions. During the later stages of the disease, severe cardiac fibrosis occurs, compromising cardiac function. Previously, our research showed that the matricellular protein CCN5 has antifibrotic properties. Therefore, we hypothesized that CCN5 gene transfer would ameliorate cardiac fibrosis and thus improve cardiac function in DMD-induced cardiomyopathy. We utilized mdx/utrn (±) haploinsufficient mice that recapitulated the DMD-disease phenotypes and used an adeno-associated virus serotype-9 viral vector for CCN5 gene transfer. We evaluated the onset of cardiac dysfunction using echocardiography and determined the experimental starting point in 13-month-old mice. Two months after CCN5 gene transfer, cardiac function was significantly enhanced, and cardiac fibrosis was ameliorated. Additionally, running performance was improved in CCN5 gene-transfected mice. Furthermore, in silico gene profiling analysis identified utrophin as a novel transcriptional target of CCN5. This was supplemented by a utrophin promoter assay and RNA-seq analysis, which confirmed that CCN5 was directly associated with utrophin expression. Our results showed that CCN5 may be a promising therapeutic molecule for DMD-induced cardiac and skeletal dysfunction.

The TSP-1 domain of the matricellular protein CCN5 is essential for its nuclear localization and anti-fibrotic function.

The matricellular protein CCN5 exerts anti-fibrotic activity in hearts partly by inducing reverse trans-differentiation of myofibroblasts (MyoFBs) to fibroblasts (FBs). CCN5 consists of three structural domains: an insulin-like growth factor binding protein (IGFBP), a von Willebrand factor type C (VWC), and a thrombospondin type 1 (TSP-1). In this study, we set out to elucidate the roles of these domains in the context of the reverse trans-differentiation of MyoFBs to FBs. First, human cardiac FBs were trans-differentiated to MyoFBs by treatment with TGF-ß; this was then reversed by treatment with recombinant human CCN5 protein or various recombinant proteins comprising individual or paired CCN5 domains. Subcellular localization of these recombinant proteins was analyzed by immunocytochemistry, cellular fractionation, and western blotting. Anti-fibrotic activity was also evaluated by examining expression of MyoFB-specific markers, α-SMA and fibronectin. Our data show that CCN5 is taken up by FBs and MyoFBs mainly via clathrin-mediated endocytosis, which is essential for the function of CCN5 during the reverse trans-differentiation of MyoFBs. Furthermore, we showed that the TSP-1 domain is essential and sufficient for endocytosis and nuclear localization of CCN5. However, the TSP-1 domain alone is not sufficient for the anti-fibrotic function of CCN5; either the IGFBP or VWC domain is needed in addition to the TSP-1 domain.