Connective Tissue Growth Factor Concentration Research Articles

Connective Tissue Growth Factor in Patients with Coronary Heart Disease: a Pilot Study

AIM. To determine the concentration of connective tissue growth factor and assess the relationship of this index with a number of pathological conditions in elderly patients with CHD. MATERIAL AND METHODS. The study enrolled 50 patients older than 75 years with a diagnosis of coronary heart disease (CAD); most of them (71%) were women. The patients ranged in age from 75 to 96 years (mean age -87.8 years). The concentration of connective tissue growth factor (CTGF) in blood was determined by enzyme immunoassay. RESULTS AND DISCUSSION. In the group of CHD patients, the average CTGF concentration was 357.2 pg/ml, whereas in healthy young adults it was 1076.7 pg/ml (p=0.07). Only 7.9% of patients with CHD had CTGF level exceeding 1000 pg/ml, whereas in young subjects CTGF level exceeded 1000 pg/ml in 25% of cases. In patients with clinically significant CHD, CTGF concentration was significantly higher than in patients without CHD (p=0.001). A negative correlation was registered between the CTGF levels and the systolic (r = -0.25; p = 0.1) and diastolic (r = -0.36; p = 0.02) blood pressure. In the subgroup of patients with pneumosclerosis, the median CTGF concentration reached 190.7 pg/l, without it – 34.7 pg/ml (p = 0.03). A significant inverse correlation was found between the CTGF levels and glucose (r = -0.34; p = 0.03), total cholesterol (r = -0.49; p = 0.002) and LDL cholesterol (r = -0.40; p = 0.01) concentrations. This pilot study found a significant effect of CTGF on echocardiographic parameters and the course of CHD, and higher levels of CTGF in patients with pneumosclerosis. However, a small sample of patients and extremely variable CTGF values do not currently allow unequivocal conclusions to be drawn at this time about the role of this factor in various comorbid conditions. CONCLUSION. Further research is needed to establish the clinical significance of CTGF.

Loss of connective tissue growth factor permits mesenchymal transition in ovarian cancer (260)

<b>Objectives:</b> Epithelial-mesenchymal transition (EMT) is a characteristic feature of ovarian cancer (OC) metastasis. Our study aimed to determine the role of Connective Tissue Growth Factor (CTGF) in EMT. <b>Methods:</b> R182 and R2615 are epithelial OC cell lines that are CK18+, b-catenin+, TWIST -, and vimentin+. Time-course experiments +/- Transforming Growth Factor-b (TGF-b) and a TGF-b inhibitor were performed to evaluate the timing of CTGF expression in EMT. R182 and R2615 CTGF knockouts (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector. Phenotypic characterization of wild types (WT) and CTGF KO cell lines were performed with the assays, including scratch wound, proliferation, and anoikis resistance assays. Scratch wound assays were treated with 50 ng/mL and 100 ng/mL concentrations of recombinant CTGF for 48 hours (hr). Cells were plated in triplicate, and confluence was measured. Proliferation was determined by cell count using Biotek Cytation over 72 hr. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive cell plate with cell growth media. Using Promega CellTiter assay, cell viability was quantified by absorbance at 490 nm at the following time points: 1hr, 24hr, 48hr, and 120hr. Western blots were performed to evaluate the expression of epithelial and mesenchymal markers. RNA sequence analysis was performed, and pathway analysis was evaluated using the iPathway guide. <b>Results:</b> TGF-b induced CTGF expression in both OC cell lines tested. CTGF was induced as early as 30 minutes post-treatment, continued to increase until 6hr, and expression was maintained until 24 hr. No CTGF expression was seen in the mesenchymal mR182 and mR2615 OC cell lines. CTGF KO was confirmed by western blot analysis and indel sequencing. CTGF KO had no effect on the expression of epithelial markers, b-catenin, CK18, and vimentin, but SNAIL, a mesenchymal marker, was induced. Loss of CTGF also had no effect on cell proliferation but promoted anoikis resistance. R182 CTGF KO cells displayed 80% viability when grown in detached conditions for 48hr compared to only 20% viability in WT cells. At 120hr, 30% of KO cells remain viable compared to <10% for WT cells. R2615 WT and KO cells demonstrated similar results. Finally, the scratch assay showed that CTGF KO cells decrease the time to confluence compared to WT. Administration of recombinant CTGF does not rescue the phenotype to WT levels.Fig. 1 <b>Conclusions:</b> CTGF is expressed early in epithelial OC cells during EMT and is regulated by TGF-b. Our data suggest that CTGF permits maintenance of an epithelial phenotype through inhibition of SNAIL expression, and loss of CTGF should be evaluated as a poor prognosis indicator in OC.

The value of plasma hypoxia markers for predicting imaging-based hypoxia in patients with head-and-neck cancers undergoing definitive chemoradiation

BackgroundTumor hypoxia worsens the prognosis of head-and-neck squamous cell carcinoma (HNSCC) patients, and plasma hypoxia markers may be used as biomarkers for radiotherapy personalization. We therefore investigated the role of the hypoxia-associated plasma proteins osteopontin, galectin-3, vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) as surrogate markers for imaging-based tumor hypoxia. MethodsSerial blood samples of HNSCC patients receiving chemoradiation within a prospective trial were analyzed for osteopontin, galectin-3, VEGF and CTGF concentrations. Tumor hypoxia was quantified in treatment weeks 0, 2 and 5 using [18F]FMISO PET/CT. The association between PET-defined hypoxia and the plasma markers was determined using Pearson’s correlation analyses. Receiver-operating characteristic analyses were conducted to reveal the diagnostic value of the hypoxia markers. ResultsBaseline osteopontin (r = 0.579, p < 0.01) and galectin-3 (r = 0.429, p < 0.05) correlated with the hypoxic subvolume (HSV) prior to radiotherapy, whereas VEGF (r = 0.196, p = 0.36) and CTGF (r = 0.314, p = 0.12) showed no association. Patients with an HSV > 1 mL in week 2 exhibited increased VEGF (p < 0.05) and CTGF (p < 0.05) levels in week 5. Pretherapeutic osteopontin levels were higher in patients exhibiting residual hypoxia at the end of treatment (104.7 vs. 60.8 ng/mL, p < 0.05) and could therefore predict residual hypoxia (AUC = 0.821, 95% CI 0.604–1.000, p < 0.05). ConclusionIn this exploratory analysis, osteopontin correlated with the initial HSV and with residual tumor hypoxia; therefore, there may be a rationale to study hypoxic modification based on osteopontin levels. However, as plasma hypoxia markers do not correspond to any spatial information of tumor hypoxia, they have limitations regarding the replacement of [18F]FMISO PET-based focal treatments. The results need to be validated in larger patient cohorts to draw definitive conclusions.

Role of transforming growth factor-beta 1 and connective tissue growth factor levels in coronavirus disease-2019-related lung Injury: a prospective, observational, cohort study.

ABSTRACTBackground: Coronavirus disease-2019 (COVID-19) results in acute lung injury. This study examined the usefulness of serum transforming growth factor-beta 1 (TGF-β1) and connective tissue growth factor (CTGF) levels in predicting disease severity in COVID-19 patients with pulmonary involvement.Methods: Fifty patients with confirmed COVID-19 and pulmonary involvement between September 2020, and February 2021 (Group 1) and 45 healthy controls (Group 2) were classified into three subgroups based on clinical severity: moderate, severe, and critical pneumonia. Serum TGF-β1 and CTGF concentrations were measured on days 1 and 7 of admission in Group 1 using an enzyme-linked immunosorbent assay. These concentrations were also measured in control cases. The mean serum TGF-β1 and CTGF levels were then compared among COVID-19 patients, based on clinical severity.Results: Significantly higher mean serum TGF-β1 and CTGF levels were observed on both days in Group 1 than in the control group. The mean serum TGF-β1 and CTGF levels on day 7 were also significantly higher than those on day 1 in Group 1. The critical patient group had the highest serum TGF-β1 and CTGF levels on both days, and the difference between this group and the moderate and severe pneumonia groups was significant. Cutoff values of 5.36 ng/mL for TGF-β1 and 626.2 pg/mL for CTGF emerged as predictors of COVID-19 with pulmonary involvement in receiver-operating characteristic curve analysis. Conclusions: TGF-β1 and CTGF are potential markers that can distinguish COVID-19 patients with pulmonary involvement and indicate disease severity. These findings may be useful for initiating treatment for early-stage COVID-19.

CONNECTIVE TISSUE GROWTH FACTOR (CTGF) AS A PROMOTER IN THE DEVELOPMENT OF FIBROSIS IN PATIENTS WITH PRIMARY BILIARY CHOLANGITIS

Primary biliary cholangitis (PBC), an immune-mediated disease, is characterized by destroying the intrahepatic bile ducts, leading to progressive damage to the biliary tree, cholestasis, and development of progressive fibrosis leading to cirrhosis with all its complications. The development of fibrosis is multifactorial and includes connective tissue growth factor (CTGF). in a mouse model of cholestasis by bile duct ligation, the hepatic and serum increase in CTGF associated with the progression of fibrosis was demonstrated. Our goal was to determine the relationship between CTGF levels and their association with the development of fibrosis in patients with PBC. Prospective, cross-sectional, and analytical study, including patients with PBC. The degree of fibrosis was determined by transient elastography (Fibroscan). Serum concentrations of FCTC-8pg/ml were quantified, for statistical analysis, the SPSS version 25.0 software was used; the medians (Q3, Q1) of CTGF, alkaline phosphatase, gamma-glutamyl-transpeptidase, and degree of fibrosis were compared with the Mann-Whitney U test with significantly less than 0.05. We included 30 patients, 29 women (96.6%) and 1 man (3.4%), with a mean age of 55.5±12.4 years. Overexpression of CTGF protein was shown in 28 subjects (93.3%). Regarding the degree of fibrosis, all patients were categorized into one of two stages: Significant fibrosis (F2) and cirrhosis (F4). The F2 group had 11 patients with a median and standard deviation for CTGF of 915.9 and 522.9, respectively; The F4 group had 19 patients who showed a median: 945.7 (1313.85-738.32); p :0.025 . In relation to the differences between fibrosis levels and markers of alkaline phosphatase cholestasis, the median and interquartile ranges F2: 79 (180.60) F4: 169(266-5.84) p : 0.066 ; GGT: F2: 1.51(7.7-1,04); F4: 1.2(2-1.92) p:0.746 In (Figure 1) The difference in medians of patients with different degrees of fibrosis and different concentration of CTGF is shown, confirming the association between peptide and the development and progression of fibrosis. According to the results obtained in patients with CBP and chronic cholestasis, the increase in CTGF showed significant differences between the degree of fibrosis and its levels; this could perhaps be interpreted as if it were an important factor for the development and progression of liver fibrosis, taking into account the antecedent of the initial study in mice with bile duct ligation and secondary cholestasis, where this factor was overexpressed at the hepatic and serum level in subjects with advanced fibrosis. It will be important to add more samples to this work and compare it with healthy controls to have better evidence. Connective tissue growth factor (CTGF) probably participates directly in the processes of synthesis of extracellular matrix and therefore in the progression of fibrosis in subjects with primary biliary cholangitis, which makes it a possibility of a therapeutic target to develop in future studies. The authors declare that there is no conflict of interest.

Diagnostic value of serum connective tissue growth factor in rheumatoid arthritis.

Serum connective tissue growth factor (CTGF) is reported to be a potential biomarker for the diagnosis of rheumatoid arthritis (RA). Our study aimed to investigate the prevalence of serum CTGF and the association with the clinical features in RA patients. Serum samples were obtained from 180 patients with RA, 168 patients with other rheumatic diseases, including 43 systemic lupus erythematosus (SLE), 34 osteoarthritis (OA), 17 primary Sjögren's syndrome (pSS), 20 ankylosing spondylitis (AS), 23 psoriatic arthritis (PsA), 6 reactive arthritis (ReA), 20 systemic sclerosis (SSc), and 5 systemic vasculitis (SV), and 64 healthy individuals. The clinical and laboratory data of patients with RA were collected. Levels of CTGF in serum were measured by enzyme linked immunosorbent assay (ELISA). Associations between CTGF and the clinical features of RA were analyzed. The positivity of serum CTGF among RA patients (33.89%) was significantly higher than those of SLE (9.3%), OA (0%), AS (0%), pSS (0%), PsA (0%), ReA (0%), SSc (5%), SV (0%), and healthy controls (4.69%) (p< 0.0001). The mean concentration of serum CTGF in RA was also significantly higher than those in other rheumatic diseases and healthy controls (p< 0.001). At the cut-off value of 263.30pg/ml, the sensitivity, specificity, positive predictive value, and negative predictive value of serum CTGF for RA were 33.89%, 96.55%, 88.41%, and 55.45%, respectively. Anti-cyclic citrullinated peptide (anti-CCP) antibody (p< 0.001), rheumatoid factor (RF) (p < 0.001), IgG (p= 0.025), and IgM (p= 0.004) in CTGF-positive patients were higher than those in CTGF-negative patients. Besides, the positive rate of serum CTGF was significantly higher in RA patients with interstitial lung disease (ILD) (53.1%, 26/49) than RA-non-ILD patients (26.7%, 35/131, p= 0.003). Serum CTGF, as a novel biomarker, has certain diagnostic value for RA. Further studies are necessary to get more knowledge for the diagnostic performance of CTGF in RA. KEY POINTS: • Serum CTGF, as a novel biomarker, has certain diagnostic value for RA, the sensitivity, specificity, positive predictive value, and negative predictive value of which were 33.89%, 96.55%, 88.41%, and 55.45%, respectively. • Serum CTGF was more common to be positive in RA-ILD patients (53.1%, 26/49) than RA-non-ILD patients (26.7%, 35/131, p= 0.003).

Effect Of Local Modulation In Enzymatic Homeostasis On Bone Turnover Marker Dynamics In Blood At Substituting Femur Defects With Vaterite Scaffolds

The goal of this research was the investigation of concentration changes in the blood bone turnover markers during local modulation of enzymatic homeostasis by means of targeted delivery of alkaline phosphatase (ALP) with polycaprolactone (PCL) and vaterite (VT) scaffolds implanted into the femur defects in white rats. Material and Methods ― The tests of PCL/VT/ALP scaffold implantations into the bone defects were performed on 30 white rats, and the serum of intact animals was used as the control. ELISA and multiplex assay were used to find inflammatory and bone turnover markers including monocyte chemoattractant-1, sclerostin, fibroblast growth factor-23, connective tissue growth factor (CTGF), osteoprotegerin, osteocalcin, β-сross laps and the activity of tartrate-resistant acid phosphatase-5b in the blood of experimental animals. The activity of serum ALP was tested with the conventional kinetic method. The morphology of the reparative processes was verified by microscopy of specimens taken from the implantation areas and stained with hematoxylin or eosin. Results ― The PCL/VT/ALP scaffold implantations into the bone defects of white rats caused active osteogenesis along with the steady rise in osteocalcin concentration in blood. ALP activity in the blood did not depend on the exogenous enzyme in the scaffold and rose by the 28th day after the implantations. The targeted ALP delivery into the defect area caused the rise in CTGF concentration as well as the decrease in blood sclerostin within a short time after the implantations. Conclusion ― The modulation of the local enzyme homeostasis by means of the targeted ALP delivery with PCL/VT scaffolds stimulated reparative osteogenesis within a short time after the implantations with no changes to the bloodstream or local inflammatory changes suggesting their biocompatibility and the safety in use.

VALUE OF CTGF IN ASSESSMENT OF LIVER GRAFT WITH NON-ALCOHOLIC FATTY LIVER DISEASE AFTER BRAIN DEATH

Background and Purpose: As the prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing globally, an accurate evaluation of liver steatosis in potential donors is required for liver transplantation. Connective tissue growth factor (CTGF) plays an important role in the acute hepatic inflammation, chronic liver injury and liver fibrosis. In this study, intrahepatic and systemic CTGF expressions were measured from brain-dead donors (BDDs) with NAFLD, and evaluated as biomarker of NAFLD-induced graft injury. Methods: Liver and blood samples were obtained from brain-dead organ donors and patients with angiomas for RNA-sequencing and validation by RT-PCR, WB and IHC. The NAFLD rat model was established by feeding rats with the high fat diet, and thereafter brain death was developed by increasing intracranial pressure in healthy or NAFLD rats. The samples from NAFLD rats, brain-dead rats and the controls were used for further detection and analysis. Systemic CTGF levels from human and rats were measured by ELISA and analyzed the relation with the quantitative histopathological score in livers from NAFLD donors. Results: Through transcriptome analysis, CTGF was down-regulated in the healthy liver from BDDs with the fold change of 0.34 (P<0.05), which was further verified in the healthy livers from humans and rats by RT-PCR, WB and IHC (P<0.05). In parallel, CTGF protein was highly overexpressed in the fatty liver compared with the healthy liver after brain death by IHC (P<0.05). The correlation analysis indicated a linear correlation between serum levels and intrahepatic expression of CTGF in both humans and rats (P<0.05). CTGF concentration correlated with the severity of NAFLD-induced liver injury. Conclusion: After brain death, CTGF was down-regulated in the healthy liver and still over-expressed in the fatty liver from donor with NAFLD. Serum CTGF level from BDDs might be a predictor of steatosis severity for graft evaluation.

Resolvin D1 suppresses pannus formation via decreasing connective tissue growth factor caused by upregulation of miRNA-146a-5p in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint stiffness, finally leading to tissue destruction. Connective tissue growth factor (CTGF) is a critical factor in RA progression, which promotes fibroblast-like synoviocyte (FLS) proliferation, pannus formation, and the damage of cartilage as well as bone. Resolvin D1 (RvD1) can promote inflammation resolution in acute inflammatory diseases, and recently, effects of RvD1 on chronic inflammatory diseases also attracted attention. This study aimed to examine the effect of RvD1 on pannus formation in RA and the underlying mechanism.MethodsSerum levels of RvD1 and CTGF were determined in RA patients and healthy persons by UPLC-MS/MS and ELISA respectively. The levels of CTGF and inflammatory factors were assessed by qRT-PCR and ELISA. MicroRNA expression profile was determined by miRNA microarray. The effects of CTGF, RvD1, and miR-146a-5p on angiogenesis were evaluated with tube formation and chick chorioallantoic membrane (CAM) assays. Collagen-induced arthritis (CIA) mice were constructed to detect the effects of RvD1 and miR146a-5p on RA. STAT3 activation was determined by Western blotting.ResultsRvD1 levels decreased while CTGF levels increased in RA patients’ serum, and an inverse correlation of the concentrations of RvD1 and CTGF in the serum of RA patients was synchronously observed. In CIA mice, RvD1 suppressed angiopoiesis and decreased the expression of CTGF. Simultaneously, RvD1 significantly decreased CTGF and pro-inflammation cytokines levels in RA FLS. Furthermore, CTGF suppressed angiopoiesis and RvD1 inhibited the proliferation and migration of RA FLS and angiopoiesis. MiRNA microarray and qRT-PCR results showed that RvD1 upregulated miRNA-146a-5p. The transfection experiments demonstrated that miRNA-146a-5p could decrease inflammatory factors and CTGF levels. Moreover, miRNA-146a-5p decreased the proliferation of FLS and angiogenesis in vivo. MiRNA-146a-5p also suppressed angiogenesis and downregulated the expression of CTGF in CIA mice. Finally, Western blot results revealed that miRNA-146a-5p inhibited the activation of STAT3.ConclusionRvD1 is prone to alleviate RA progression through the upregulation of miRNA-146a-5p to suppress the expression of CTGF and inflammatory mediators, thereby decreasing pannus formation and cartilage damage.

Evaluation of tear and aqueous humor level, and genetic variants of connective tissue growth factor as biomarkers for early detection of pseudoexfoliation syndrome/glaucoma

Pseudoexfoliation syndrome (PEX) may lead to the development of pseudoexfoliative glaucoma (PEG), a potential cause of irreversible blindness, if left untreated. This type of glaucoma often presents with much higher intraocular pressure (IOP) values than observed in primary open angle glaucoma, and patients are often unaware of their condition. Therefore, early diagnosis is of utmost importance in PEX and PEG. Unfortunately, no valid objective biomarkers are available that can be used for this purpose. The excessive synthesis and deposition of elastic microfibrillar pseudoexfoliation material is observed in the pathophysiology of PEX, therefore, growth factors may play roles in this pathology. Thus, in this study, we sought to determine the roles of phenotypes and genotypes of connective tissue growth factor (CTGF) as objective biomarkers for early diagnosis of PEX and PEG. Thus, we investigated possible associations involving tear and aqueous humor CTGF concentrations and four single nucleotide polymorphisms (SNPs) of the CTGF gene in PEX and PEG. The study was designed as a 2-year case-control study in the Turkish population. Study population was composed of 214 patients with PEG, 214 patients with PEX, and 214 age-matched controls for CTGF SNP analysis. Tear fluid study group consisted of 78 patients with PEG, 77 patients with PEX, and 78 controls. Aqueous humor analysis included 8 patients with PEG, 17 patients with PEX, and 23 controls. Tear fluid was collected using Schirmer strips, and aqueous humor samples were taken during cataract surgery. CTGF concentration was determined by ELISA, and total protein concentration was determined by Bradford assay in tear and aqueous humor samples. PCR followed by restriction fragment length polymorphism analysis was used for genotyping of rs6918698 G/C and rs9399005 C/T, while real-time PCR was used for rs9402373 C/G and rs12526196 T/C. Intraocular pressure, visual field score, mean deviation, and pattern standard deviation parameters were also evaluated. CTGF concentration in tear fluid was significantly higher in PEG patients compared with controls (P = 0.001), while it was lower in PEX patients. Similarly, total protein concentration in tear fluid was significantly increased in PEG patients relative to PEX patients (P = 0.026) and controls (P = 0.004). CTGF concentration in aqueous humor did not differ markedly between the groups, whereas total protein was significantly higher in the PEG group compared with the PEX group (P = 0.012) and controls (P = 0.003). Receiver operating characteristic analysis revealed that total protein in aqueous humor was a robust classifier for evaluating the presence of PEG against controls (Area under the curve = 0.897, P = 0.001). The genotypes of the studied SNPs were not significantly correlated with CTGF concentration in aqueous humor or tear fluid, and did not exhibit significant association with PEG or PEX. In conclusion, this was the first study to investigate tear fluid CTGF concentration in PEX and PEG, which came out not to be a good classifier for PEG or PEX. Total protein level in tear fluid and CTGF SNPs also did not predict PEG or PEX status successfully.

Influence of fibrosis mediators on the outcomes of endoscopic endonasal dacryocystorhinostomy

Fibrosis is the most important pathologic condition involved in undesirable outcomes of dacryocystorhinostomy. A number of biochemical factors are currently known to have an effect on wound healing by promoting excessive scarring. Isoforms of transforming growth factor β (TGF-β1) are considered the 'main' pro-fibrotic factor, but wound healing is also affected by other cytokines such as connective tissue growth factor (CTGF), which stimulates fibrosis, and fibroblast growth factor (FGF-2), which acts as antagonist to it. To investigate correlations between endoscopic endonasal dacryocystorhinostomy outcomes and certain mediators of fibrosis. The study included 45 cases of endoscopic endonasal dacryocystorhinostomy. The patients were grouped according to surgery outcome: patients with unsuccessful surgical treatment were assigned to group 1 (n=10); patients with successful surgical treatment - to group 2 (n=34). One patient was excluded from the study. Full-layer biopsy specimen were taken from patients' nasal mucosa before the surgery. TGF-β1, TGF-β2, TGF-β3, CTGF, FGF-2 concentrations were evaluated using ELISA and normalized by total protein concentration. Surgical failure was observed in 10 cases (22.72%). CTGF concentration was significantly correlated with negative outcome (p<0.05) and was elevated in most specimen obtained from group 1. No significant correlation was noted between the concentrations of other evaluated cytokines in nasal mucosa specimens and the surgical outcome. The study found a correlation between CTGF concentration in nasal mucosa and dacryocystorhinostomy outcome, which supports the hypothesis suggested by several authors linking dacryocystorhinostomy failure with chronic inflammation in nasal mucosa.

Is there any relation between connective tissue growth factor and scar tissue in vesicoureteral reflux?

Gültekin ND, Benzer M, Tekin-Neijmann Ş. Is there any relation between connective tissue growth factor and scar tissue in vesicoureteral reflux. Turk J Pediatr 2019; 61: 71-78. Vesicoureteral reflux (VUR) is the most common uropathy in childhood which leads to increased frequency of urinary tract infection (UTI) and renal scarring. Connective tissue growth factor (CTGF) plays an important role in the development of glomerular and tubulointerstitial fibrosis in progressive kidney diseases. The aim of this study was to investigate the relation between urinary CTGF and renal damage resulted from VUR. This cross sectional study included 70 patients with VUR and 62 healthy sex and age matched children. Urinary creatinine and CTGF (uCTGF) concentrations were analysed in all cases and CTGF to creatinine ratio were calculated. The records of radiologic evaluations of the patients including ultrasound, voiding cystouretrography and 99m-technetium dimercaptosuccinic acid (DMSA) scintigraphy were obtained retrospectively. The patient group was further divided into two groups according to the existence of renal cortical scarring in the DMSA scan. The study consisted of three groups; Group 1 (control group) 62 children, Group 2 (VUR positive, scar negative) 24 patient, Group 3 (VUR positive, scar positive) 46 patient (VUR+scar). The medians of uCTGF and uCTGF to creatinine ratio of the three groups were significantly different (p < 0.001). Pairwise group comparisons revealed that Group 1 had significantly lower uCTGF level and uCTGF/creatinine ratio, as compared to Groups 2 and 3 (p < 0.001 and p=0.002, respectively). There was no statistically significant difference between Groups 2 and 3 (p=0.052). uCTGF is significantly increased in children with VUR, independent on the presence of renal scarring. Increased uCTGF, even in the absence of the renal scarring, could be interpreted as development and a progression of glomerular and tubulointerstitial fibrosis in vesicoureteral reflux. Further experimental and clinical investigations are required to fully elucidate the mechanism of CTGF in vesicoureteral reflux.

Effects of connective tissue growth factor on prostate cancer bone metastasis and osteoblast differentiation.

Previous studies have demonstrated that connective tissue growth factor (CTGF) is expressed at increased levels in prostate cancer bone metastasis mouse models and patients with prostate cancer which metastasizes to the bone; however, the underlying molecular mechanism(s) remain unknown. The present study investigated the function of CTGF in osteoblast differentiation and its effect on prostate cancer bone metastasis by analyzing CTGF gene expression and transcription at different levels of invasion, metastasis of prostate cancer cells, and the influence of CTGF on proliferation and xenotransplantation. A mouse model demonstrating bone metastasis was used to investigate the function(s) of CTGF in bone metastasis and osteoblast differentiation. Results demonstrated that CTGF expression was increased in association with high bone metastasis in prostate cancer cells, and its expression was significantly decreased in whole cell lysates. CTGF expression in prostate cancer cells with high levels of bone metastasis was increased 1.9-fold compared with prostate cancer cells with low levels of bone metastasis. The expression of CTGF in mesenchymal cells was markedly increased compared with epithelial cells. Results indicated that the increased expression of CTGF does not affect the proliferation of tumor cells and possesses no influence on tumor volume. Control and CTGF plasmids were transfected into RM1 cells and led to 4 and 17% bone lesions, respectively. Increased expression of CTGF significantly enlarged the tumor area in the bone metastatic position compared with the control. Positive areas of alkaline phosphatase were significantly decreased as the concentration of CTGF increased. The results of the present study demonstrated that CTGF promotes prostate carcinoma to metastasize in the bone by dysregulating osteoblast differentiation.

Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies.

Serum connective tissue growth factor is a highly discriminatory biomarker for the diagnosis of rheumatoid arthritis

BackgroundOur previous proteomic study indicated that connective tissue growth factor (CTGF) may be a potential biomarker for rheumatoid arthritis (RA) diagnosis. The aim was to assess the performance of CTGF as a biomarker of RA.MethodSerum and synovial fluid CTGF was detected using a direct high sensitivity sandwich ELISA kit. Serum CTGF levels were tested for discriminatory capacity and optimal assay cutoffs determined in a training cohort of 98 cases of RA with 103 healthy controls. The assay performance was then validated in a further cohort of 572 patients (with RA (n = 217), ankylosing spondylitis (n = 92), gout (n = 74), osteoarthritis (n = 52), systemic lupus erythematosus (n = 72), or primary Sjögren’s syndrome (pSS) (n = 65)).ResultsSignificant elevation of synovial fluid CTGF concentration was found in RA patients, demonstrating excellent diagnostic ability to predict RA (area under the curve (AUC) = 0.97). Similar results were found in serum CTGF detection. At the optimal cutoff value 88.66 pg/mL, the sensitivity, specificity, and the AUC was 0.86, 0.92, and 0.92, respectively, in the training cohort. Similar performance was observed in the validation cohort, with sensitivity, specificity, positive likelihood, and negative likelihood of 0.82, 0.91, 5.74, and 0.12, respectively. Stronger discriminatory capacity was seen with the combination of CTGF and anti-citrullinated protein antibody (ACPA) (AUC = 0.96) than with either ACPA or rheumatoid factor (RF) alone (AUC = 0.80 or 0.79, respectively). The discriminatory performance of serum CTGF was consistent across all inflammatory conditions tested (AUC >0.92 in all cases), with the sole exception of pSS. Serum CTGF did not vary with symptom duration or disease activity.ConclusionsSerum CTGF is a promising diagnostic biomarker for RA, with performance in the current study better than either ACPA or RF.

Effects of connective tissue growth factor on human periodontal ligament fibroblasts

ObjectiveThe aim of this study was to evaluate the effects of different concentrations of connective tissue growth factor (CTGF) on human periodontal ligament fibroblasts(HPLFs). DesignHPLFs were cultured and identified. Then, different concentrations of CTGF (1, 5, 10, 50, 100ng/ml) were added to the HPLF culture. Next, CCK-8 assays, alkaline phosphatase (ALP) assays, hydroxyproline determination, alizarin red staining methods, Transwell chambers and real-time PCR methods were applied to observe the effects of CTGF on the proliferation, ALP activity, synthesis of collagen, formation of mineralized nodules and migration. We also studied expression of ALP, fiber link protein (FN), integrin-binding sialoprotein (IBSP), osteocalcin (OC), and integrin beta 1 (ITGB1) mRNA by HPLFs. Statistical significance was assumed if P<0.05 or P<0.01. ResultsThe addition of CTGF (1, 5, 10ng/ml) remarkably promoted the proliferation and collagen synthesis of HPLFs compared with controls. CTGF (1, 5, 10, 50ng/ml) improved ALP activity of HPLFs, and at all concentrations, CTGF (1, 5, 10, 50, 100ng/ml) improved the expression of ALP, FN, IBSP and ITGB1 mRNA. In addition, CTGF (1, 5, 10, 50, 100ng/ml) promoted the migration of HPLFs, which was dose-dependent, with maximal promotion in the 10ng/ml group (P<0.05 or P<0.01). ConclusionsThus, in a certain range of concentrations, CTGF can promote the biological effects, including proliferation, migration and collagen synthesis of HPLFs, to promote the differentiation of HPLFs in the process of osteogenesis.

Hypertrophy and Fibrosis of the Ligamentum Flavum in Lumbar Spinal Stenosis is Associated With Increased Expression of LPA and LPAR1.

Histologic, immunohistochemical (IHC), and enzyme-linked immunosorbent assay analysis of the human ligamentum flavum (LF). The purpose of this study was to determine the level of expression of lysophosphatidic acid (LPA) in the LF from lumbar spinal stenosis (LSS) patients and to analyze the relationship among LPA, LPA receptors (LPARs), and LF hypertrophy. LF hypertrophy and fibrosis are important causes of LSS. LPA is involved in the fibrotic process in multiple organ systems. Therefore, we hypothesized that LPA and its receptors might also play a role in degeneration of the LF in LSS patients. Forty-one LF samples were enrolled in this study. The thickness of the LF was measured by magnetic resonance imaging. Histologic analysis using hematoxylin and eosin and Masson trichrome stain was performed for each LF to evaluate the architecture of the extracellular matrix. The content of LPA and connective tissue growth factor (CTGF) in LF samples was analyzed using enzyme-linked immunosorbent assay. The expression of LPAR1 was determined by IHC. Degeneration of the LF was characterized by an increase in disorganized elastic fibers and fibrotic transformation by extracellular collagen deposition. The thickness of the LF and the concentration of LPA and CTGF in the hypertrophic LF group were significantly higher than the control group. Furthermore, the LPA and CTGF concentrations had a positive correlation with the LF thickness (r=0.91, P<0.001 and r=0.943, P<0.001, respectively). On the basis of IHC analysis, the expression of LPAR1 was increased in the hypertrophy group. The increased expression of LPA and LPAR1 is associated with the fibrosis and hypertrophy of the LF in patients with LSS. Further study on the mechanism underlying LF fibrosis may lead to new therapies for LF hypertrophy and fibrosis.

The effect of connective tissue growth factor (CTGF) and CTGF antibody on collagen secretion of chicken tendon synovial cells

Objective To investigate the effect of CTGF and CTGF antibody in different concentration on collagen secretion of tendon synovial cells. Methods From February, 2014 to August, 2014, tendon synovial cells were obtained from LegHorn chicken tendon synovial tissues and cultured in Petri dish with incremental concentration of CTGF or CTGF antibody, ELISA was used to detect secretion of collagen Ⅰ/Ⅲ. Results The secretion of collagenⅠincreased gradually as the concentration of CTGF elevating from 5 ng/ml to 50 ng/ml, there were significant differences between the groups (P < 0.05) . While the secretion of collagen Ⅲincreased gradually as the concentration of CTGF elevating from 10 ng/ml to 200 ng/ml, no significant difference was observed between the other groups. CollagenⅠof synovial cells showed a downward trend on secretion when cultured with CTGF antibody as the concentration elevating from 8 μg/ml to 32 μg/ml, there were significant differences between the groups (P < 0.05) . While the secretion of collagen Ⅲ showed a downward trend when cultured with CTGF antibody of the concentration 4 μg/ml, there was no significant difference between the other groups. Conclusion CTGF can promote collagen secretion of synovial cells in vitro with an optimal concentration, the concentration was 50 ng/ml (collagen I) , 200 ng/ml (collagen III) . CTGF antibody can inhibit collagen secretion of synovial cell. Key words: Connective tissue growth factor; Synovial cells; Collagen; Chicken

FG-3019, a Human Monoclonal Antibody Recognizing Connective Tissue Growth Factor, is Subject to Target-Mediated Drug Disposition.

PurposeTo evaluate and model the pharmacokinetic and pharmacodynamic behavior in rats of FG-3019, a human monoclonal antibody targeting connective tissue growth factor (CTGF).MethodsFG-3019, human CTGF (rhCTGF), or the N-terminal domain of rhCTGF were administered intravenously to rats and concentrations of these proteins as well as endogenous CTGF were determined by immunoassays. FG-3019, or 125I-labeled FG-3019, and human CTGF (rhCTGF) were co-administered to assess the impact of CTGF on the elimination rate and tissue localization of FG-3019, which was further characterized by immunohistochemical analysis. A PK/PD model for target-mediated elimination of FG-3019 was developed to fit the kinetic data.ResultsFG-3019 exhibited non-linear pharmacokinetics in rats. Circulating concentrations of the N-terminal half of CTGF increased after dosing with FG-3019, reached maximal levels after 1–5 days, and returned toward baseline levels as FG-3019 cleared from the circulation, whereas the concentration of intact CTGF was unaffected by administration of FG-3019. Co-administration of rhCTGF dramatically enhanced the rate of FG-3019 elimination, redistributing the majority of 125I-labeled FG-3019 from the blood to the liver, kidney, spleen and adrenal gland. FG-3019 co-administered with CTGF was found along the sinusoids of the liver and adrenal glands, the capillaries of the kidney glomeruli and in the spleen. A pharmacokinetic model for target-mediated elimination of FG-3019 was used to fit the time courses of FG-3019 and endogenous CTGF plasma concentrations, as well as time courses of rhCTGF and rhCTGF N-fragment after intravenous administration of these species.ConclusionsFG-3019 is subject to target mediated elimination in rats.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-016-1918-0) contains supplementary material, which is available to authorized users.

Angiotensin-(1-7) Attenuates Skeletal Muscle Fibrosis and Stiffening in a Mouse Model of Extremity Sarcoma Radiation Therapy

Radiation-induced fibrosis (RIF) of musculoskeletal tissue is a common complication of radiation therapy for extremity soft-tissue sarcoma, with no standardized strategy for prevention and treatment. Angiotensin-(1-7) (Ang-[1-7]), a well-tolerated endogenous heptapeptide hormone with antitumor and antifibrotic properties, was tested as a radioprotectant for RIF and stiffening of irradiated muscles. Male CD-1 mice were randomized to one of three treatment groups: control, simulated sarcoma radiation therapy to the gastrocnemius and soleus muscles, or radiation therapy along with continuous Ang-(1-7) delivery initiated three days before radiation therapy. The biologically equivalent dose of radiation (∼100.3 Gy) absorbed by normal musculature during the course of radiation therapy for extremity sarcoma was delivered by means of four dose fractions of 7.3 Gy over two weeks. Fibrosis (n = 5 per group) and mechanical properties (n = 4 to 6 per group) of the muscles were measured at six weeks and four months after radiation therapy, and the intramuscular concentration of the profibrotic cytokines transforming growth factor-beta (TGF-β) and connective tissue growth factor (CTGF) (n = 8 to 10 per group) were measured at six weeks. Interstitial (p < 0.01) and perivascular (p < 0.05) fibrosis increased significantly in the muscles treated with radiation therapy alone versus the nonirradiated controls at both six weeks (interstitial, +89%; perivascular, +112%) and four months (interstitial, +154%; perivascular, +88%). The muscles treated with radiation alone also exhibited increased tension (p < 0.01) versus nonirradiated controls at both six weeks (+779%) and four months (+1761%) when placed under 5% strain, and at four months (+1390%; p < 0.001) under 10% strain. At four months, muscle stiffness had increased in the mice treated with radiation therapy alone (+90%; p = 0.002) compared with nonirradiated controls. TGF-β production was also greater in this group at six weeks (+37%; p = 0.06) versus control. Ang-(1-7) administration prevented RIF and stiffening, with no differences observed for any other outcome between those receiving radiation therapy with Ang-(1-7) and the nonirradiated controls. Likewise, Ang-(1-7) mitigated the increase in TGF-β and CTGF concentration from radiation therapy. Ang-(1-7) attenuated RIF, stiffening, and production of profibrotic cytokines that were elevated in mouse skeletal muscles after simulated radiation therapy for extremity sarcoma. Ang-(1-7) may serve as a potential therapy for the prevention of RIF in patients who require radiation therapy as adjuvant treatment for soft-tissue sarcoma.