CCN2-induced lymphangiogenesis is mediated by the integrin αvß5-ERK pathway and regulated by DUSP6.

Lymphangiogenesis is essential for the development of the lymphatic system and is important for physiological processes such as homeostasis, metabolism and immunity. Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated the effects of CCN2 on lymphangiogenesis. In in vivo Matrigel plug assays, exogenous CCN2 increased the number of Podoplanin-positive vessels. Subsequently, we found that CCN2 induced phosphorylation of ERK in primary cultured LECs, which was almost completely inhibited by the blockade of integrin αvß5 and partially decreased by the blockade of integrin αvß3. CCN2 promoted direct binding of ERK to dual-specific phosphatase 6 (DUSP6), which regulated the activation of excess ERK by dephosphorylating ERK. In vitro, CCN2 promoted tube formation in LECs, while suppression of Dusp6 further increased tube formation. In vivo, immunohistochemistry also detected ERK phosphorylation and DUSP6 expression in Podoplanin-positive cells on CCN2-supplemented Matrigel. These results indicated that CCN2 promotes lymphangiogenesis by enhancing integrin αvß5-mediated phosphorylation of ERK and demonstrated that DUSP6 is a negative regulator of excessive lymphangiogenesis by CCN2.

Serum and glucocorticoid-regulated kinase 1 regulates transforming growth factor ß1-connective tissue growth factor pathway in chronic rhinosinusitis.

PURPOSE: Serum/glucocorticoid-regulated kinase 1 (SGK1) has been identified as a crucial regulator in fibrotic disorders. Herein, we explored SGK1 role in tissue remodeling of chronic rhinosinusitis (CRS). METHODS: Lentivirus was employed to generate an SGK1-overexpressing human bronchial epithelial cell (16HBE) line. To screen SGK1 downstream genes, RNA sequencing was performed on SGK1-overexpressing and control cell lines. To determine protein and gene expression levels, immunohistochemistry, western blotting, and quantitative real-time polymerase chain reaction were employed. Correlation analysis was performed using mRNA expression levels of SGK1, transforming growth factor ß1 (TGF-ß1), and connective tissue growth factor (CTGF) derived from CRS mucosal tissue and GEO database. Gene set enrichment analysis was conducted using gene sets from Molecular Signatures Database. The severity of symptoms in CRS patients was assessed using the 22-Item Sinonasal Outcome Test. RESULTS: SGK1 overexpression significantly increased the expression of connective tissue growth factor (CTGF) in 16HBE cells (P < 0.01). Consistently, CTGF protein level was considerably greater in mucosal tissue of CRS without nasal polyps (CRSsNP) than in CRS with nasal polyps (CRSwNP) (P < 0.05) or in control subjects (P < 0.01). TGF-ß1 protein level was higher in mucosal tissue of CRSsNP patients than in CRSwNP patients (P < 0.001) or in the control group (P < 0.01). mRNA levels of SGK1 and CTGF (P < 0.05, r = 0.668; P = 0.001, r = 0.630), TGF-ß1 and CTGF (P < 0.05, r = 0.560; P < 0.05, r = 0.420), as well as SGK1 and TGF-ß1(P < 0.05, r = 0.612; P < 0.05, r = 0.524) were significantly correlated in CRS mucosal tissue and GSE36830 dataset, respectively. TGF-ß1-induced upregulated genes were significantly enriched in SGK1 overexpression group. In vitro assays, TGF-ß1 promoted SGK1 and CTGF expression in a concentration- and time-dependent manner. Administrating an SGK1 inhibitor, GSK650394, significantly inhibited TGF-ß1-induced CTGF expression in 16HBE and dispersed primary nasal polyp cells. CONCLUSIONS: TGF-ß1 stimulation significantly increases SGK1 and CTGF expression. By regulating TGF-ß1-CTGF pathway, SGK1 may participate in tissue remodeling in the pathological mechanism of CRS.

CCN2/CTGF promotor activity in the developing and adult mouse eye.

CCN2/CTGF is a matricellular protein that is known to enhance transforming growth factor-ß signaling and to induce a myofibroblast-like phenotype in a variety of cell types. Here, we investigated Ccn2/Ctgf promotor activity during development and in the adult mouse eye, using CTGFLacZ/+ mice in which the ß-galactosidase reporter gene LacZ had been inserted into the open reading frame of Ccn2/Ctgf. Promotor activity was assessed by staining for ß-galactosidase activity and by immunolabeling using antibodies against ß-galactosidase. Co-immunostaining using antibodies against glutamine synthetase, glial fibrillary acidic protein, choline acetyltransferase, and CD31 was applied to identify specific cell types. Ccn2/Ctgf promotor activity was intense in neural crest-derived cells differentiating to corneal stroma and endothelium, and to the stroma of choroid, iris, ciliary body, and the trabecular meshwork during development. In the adult eye, a persistent and very strong promotor activity was present in the trabecular meshwork outflow pathways. In addition, endothelial cells of Schlemm's canal, and of retinal and choroidal vessels, retinal astrocytes, Müller glia, and starburst amacrine cells were stained. Very strong promoter activity was seen in the astrocytes of the glial lamina at the optic nerve head. We conclude that CCN2/CTGF signaling is involved in the processes that govern neural crest morphogenesis during ocular development. In the adult eye, CCN2/CTGF likely plays an important role for the trabecular meshwork outflow pathways and the glial lamina of the optic nerve head.

Identification of drivers of breast cancer invasion by secretome analysis: insight into CTGF signaling.

An altered consistency of tumor microenvironment facilitates the progression of the tumor towards metastasis. Here we combine data from secretome and proteome analysis using mass spectrometry with microarray data from mesenchymal transformed breast cancer cells (MCF-7-EMT) to elucidate the drivers of epithelial-mesenchymal transition (EMT) and cell invasion. Suppression of connective tissue growth factor (CTGF) reduced invasion in 2D and 3D invasion assays and expression of transforming growth factor-beta-induced protein ig-h3 (TGFBI), Zinc finger E-box-binding homeobox 1 (ZEB1) and lysyl oxidase (LOX), while the adhesion of cell-extracellular matrix (ECM) in mesenchymal transformed breast cancer cells is increased. In contrast, an enhanced expression of CTGF leads to an increased 3D invasion, expression of fibronectin 1 (FN1), secreted protein acidic and cysteine rich (SPARC) and CD44 and a reduced cell ECM adhesion. Gonadotropin-releasing hormone (GnRH) agonist Triptorelin reduces CTGF expression in a Ras homolog family member A (RhoA)-dependent manner. Our results suggest that CTGF drives breast cancer cell invasion in vitro and therefore could be an attractive therapeutic target for drug development to prevent the spread of breast cancer.

Baicalein alleviated TGF ß1-induced type I collagen production in lung fibroblasts via downregulation of connective tissue growth factor.

Although we have reported that baicalein ameliorated bleomycin-induced pulmonary fibrosis in rats and inhibited fibroblast-to-myofibroblast differentiation, the mechanisms of the capability of baicalein to suppress the production of type I collagen in fibroblasts remains unclear. Here, we showed that baicalein suppressed transforming growth factor ß1 (TGF ß1)-stimulated the production of type I collagen in lung fibroblast MRC-5 cells. By applying SILAC-based proteomic technology, 158 proteins were identified as baicalein-modulated proteins in TGF ß1-stimulated the accumulation of type I collagen in MRC-5 cells. Our proteomic and biochemical analysis demonstrated that baicalein decreased the expression levels of connective tissue growth factor (CTGF) in TGF ß1-stimulated MRC-5 cells. In addition, CTGF overexpression elevated the levels of type I collagen in baicalein-treated fibroblasts. Moreover, our results demonstrated that baicalein-downregulated CTGF expression might be related with the decrease of Smad2 phosphorylation, but not SP1. This work not only linked CTGF to TGF ß1-stimulated the production of type I collagen in its attribution to the effects of baicalein, but also might provide valuable information for enhancing the knowledge of the pharmacological inhibition of collagen production, which might represent a promising strategy for the treatment of pulmonary fibrosis.

Mice Lacking Connective Tissue Growth Factor in the Forebrain Exhibit Delayed Seizure Response, Reduced C-Fos Expression and Different Microglial Phenotype Following Acute PTZ Injection.

Connective tissue growth factor (CTGF) plays important roles in the development and regeneration of the connective tissue, yet its function in the nervous system is still not clear. CTGF is expressed in some distinct regions of the brain, including the dorsal endopiriform nucleus (DEPN) which has been recognized as an epileptogenic zone. We generated a forebrain-specific Ctgf knockout (FbCtgf KO) mouse line in which the expression of Ctgf in the DEPN is eliminated. In this study, we adopted a pentylenetetrazole (PTZ)-induced seizure model and found similar severity and latencies to death between FbCtgf KO and WT mice. Interestingly, there was a delay in the seizure reactions in the mutant mice. We further observed reduced c-fos expression subsequent to PTZ treatment in the KO mice, especially in the hippocampus. While the densities of astrocytes and microglia in the hippocampus were kept constant after acute PTZ treatment, microglial morphology was different between genotypes. Our present study demonstrated that in the FbCtgf KO mice, PTZ failed to increase neuronal activity and microglial response in the hippocampus. Our results suggested that inhibition of Ctgf function may have a therapeutic potential in preventing the pathophysiology of epilepsy.

Epithelial Vasopressin Type-2 Receptors Regulate Myofibroblasts by a YAP-CCN2-Dependent Mechanism in Polycystic Kidney Disease.

BACKGROUND: Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. METHODS: We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). RESULTS: V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. CONCLUSIONS: Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.

Insights into Fibroblast Plasticity: Cellular Communication Network 2 Is Required for Activation of Cancer-Associated Fibroblasts in a Murine Model of Melanoma.

Tumor stroma resembles a fibrotic microenvironment, being characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs). In wild-type mice injected with melanoma cells, we show that the stem cell transcription factor Sox2 is expressed by tumor cells and induced in CAFs derived from synthetic fibroblasts. These fibroblasts were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase under the control of a fibroblast-specific promoter/enhancer. Conversely, fibroblast activation was impaired in mice with a fibroblast-specific deletion of cellular communication network 2 (Ccn2), associated with reduced expression of α-smooth muscle actin and Sox2. Multipotent Sox2-expressing skin-derived precursor (SKP) spheroids were cultured from murine back skin. Using lineage tracing and flow cytometry, approximately 40% of SKPs were found to be derived from type I collagen-lineage cells and acquired multipotency in culture. Inhibition of mechanotransduction pathways prevented myofibroblast differentiation of SKPs and expression of Ccn2. In SKPs deleted for Ccn2, differentiation into a myofibroblast, but not an adipocyte or neuronal phenotype, was also impaired. In human melanoma, CCN2 expression was associated with a profibrotic integrin alpha (ITGA) 11-expressing subset of CAFs that negatively associated with survival. These results suggest that synthetic dermal fibroblasts are plastic, and that CCN2 is required for the differentiation of dermal progenitor cells into a myofibroblast/CAF phenotype and is, therefore, a therapeutic target in melanoma.

Connective Tissue Growth Factor and Renal Fibrosis.

CCN2, also known as connective tissue growth factor (CTGF), is one of important members of the CCN family. Generally, CTGF expresses at low levels in normal adult kidney, while increases significantly in various kidney diseases, playing an important role in the development of glomerular and tubulointerstitial fibrosis in progressive kidney diseases. CTGF is involved in cell proliferation, migration, and differentiation and can promote the progression of fibrosis directly or act as a downstream factor of transforming growth factor ß (TGF-ß). CTGF also regulates the expression and activity of TGF-ß and bone morphogenetic protein (BMP), thereby playing an important role in the process of kidney repair. In patients with chronic kidney disease, elevated plasma CTGF is an independent risk factor for progression to end-stage renal disease and is closely related to glomerular filtration rate. Therefore, CTGF may be a potential biological marker of kidney fibrosis, but more clinical studies are needed to confirm this view. This section briefly describes the role and molecular mechanisms of CTGF in renal fibrosis and also discusses the potential value of targeting CCN2 for the treatment of renal fibrosis.

[Proliferation effect of ligamentum flavum cells induced by transforming growth factor ß 1 and its effect on connective tissue growth factor].

OBJECTIVE: To investigate the effect of transforming growth factor ß 1 (TGF-ß 1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression. METHODS: The ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-ß 1, 50 ng/mL CTGF, 3 ng/mL TGF-ß 1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-ß 1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes. RESULTS: The morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance ( A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different ( P<0.05), there was no significant difference in A value between the cells of each generation at the same time point ( P>0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E ( P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E ( P<0.05). There were also significant differences among groups A, C and groups B, D ( P<0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E ( P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E ( P<0.05), and the difference between groups A, C and groups B, D was also significant ( P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E ( P<0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E ( P<0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E ( P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E ( P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E ( P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E ( P>0.05). CONCLUSION: TGF-ß 1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-ß 1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.

Extracellular Matrix-Associated Factors Play Critical Roles in Regulating Pancreatic ß-Cell Proliferation and Survival.

This review describes formation of the islet basement membrane and the function of extracellular matrix (ECM) components in ß-cell proliferation and survival. Implications for islet transplantation are discussed. The insulin-producing ß-cell is key for maintaining glucose homeostasis. The islet microenvironment greatly influences ß-cell survival and proliferation. Within the islet, ß-cells contact the ECM, which is deposited primarily by intraislet endothelial cells, and this interaction has been shown to modulate proliferation and survival. ECM-localized growth factors, such as vascular endothelial growth factor and cellular communication network 2, signal through specific receptors and integrins on the ß-cell surface. Further understanding of how the ECM functions to influence ß-cell proliferation and survival will provide targets for enhancing functional ß-cell mass for the treatment of diabetes.

Roles of matricellular CCN2 deposited by osteocytes in osteoclastogenesis and osteoblast differentiation.

In this study, we investigated the effect of CCN2 (cellular communication network factor 2), previously termed connective tissue growth factor, deposited in bone matrix on osteoclastogenesis and osteoblast differentiation. To mimic the bone matrix environment, osteocytic MLO-Y4 cells had been embedded in collagen-gel with recombinant CCN2 (rCCN2), and mouse macrophage-like RAW264.7 cells were inoculated on the gel and treated with receptor activator of NF-κB ligand (RANKL). NFATc1 and cathepsin K (CTSK) productions were more increased in the combination of RAW264.7 and MLO-Y4 cells treated with rCCN2 than the combination without rCCN2. Next, we isolated an osteocyte-enriched population of cells and osteoclast progenitor cells from wild type and tamoxifen-inducible Ccn2-deficient (KO) mice and performed similar analysis. NFATc1 and CTSK productions were decreased in the KO osteocyte-enriched population at 6 months after the tamoxifen injection, regardless of the origin of the osteoclast progenitor cells. Interestingly, CTSK production was rather increased in KO osteocytes at 1 year after the injection. Finally, the combination of osteoblastic MC3T3-E1 and MLO-Y4 cells in rCCN2-containing bone matrix revealed the up-regulation of osteoblastic marker genes. These findings suggest that CCN2 supplied by osteocytes regulates both osteoclastogenesis and osteoblast differentiation.

[Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factor ß 1/connective tissue growth factor].

OBJECTIVE: To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor ß 1 (TGF-ß 1)/connective tissue growth factor (CTGF). METHODS: The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-ß 1, and p38 siRNA+3 ng/mL TGF-ß 1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. RESULTS: p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ ( P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly ( P<0.05), while those in groups C and D increased significantly ( P<0.05); and those indicators significantly increased in group C than in group D ( P<0.05). CONCLUSION: TGF-ß 1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.

CTGF Triggers Rat Astrocyte Activation and Astrocyte-Mediated Inflammatory Response in Culture Conditions.

To improve clinical outcomes for patients with traumatic brain injury (TBI), it is necessary to explore the mechanism of traumatic brain injury (TBI)-induced neuroinflammation. Connective tissue growth factors (CTGF) have been reported to be involved in the process of inflammatory response or tissue repair, whereas whether and how CTGF participates in the astrocyte-mediated inflammation after TBI remains unclear. In the present study, the TBI-induced activation of astrocytes and augmentation of inflammatory response were simulated by stimulating rat astrocytes with TGF-ß1 or CTGF in cultured conditions. TGF-ß1 and CTGF both upregulated the expression of GFAP in astrocytes and facilitated the production of inflammatory cytokines and chemokines. Activation of astrocytes by CTGF is in an autocrine manner. According to the results of Boyden chamber assay, CTGF enhanced the recruitment of peripheral blood mononuclear cells (PBMCs) by reactive astrocytes. Besides, CTGF-mediated activation of astrocytes and augmentation of inflammatory response can be terminated by the inhibitor of ASK1 or p38 and JNK. Thus, our data suggested that CTGF could activate astrocytes in an autocrine manner and promote astrocyte-mediated inflammatory response by triggering the ASK1-p38/JNK-NF-κB/AP-1 pathways in astrocytes. Collectively, our study provided evidence that astrocyte-secreted CTGF serves as an amplifier of neuroinflammatory and could be a potential target for alleviating TBI-induced inflammation.

Blocking CCN2 Reduces Progression of Sensorimotor Declines and Fibrosis in a Rat Model of Chronic Repetitive Overuse.

Fibrosis may be a key factor in sensorimotor dysfunction in patients with chronic overuse-induced musculoskeletal disorders. Using a clinically relevant rodent model, in which performance of a high demand handle-pulling task induces tissue fibrosis and sensorimotor declines, we pharmacologically blocked cellular communication network factor 2 (CCN2; connective tissue growth factor) with the goal of reducing the progression of these changes. Young adult, female Sprague-Dawley rats were shaped to learn to pull at high force levels (10 min/day, 5 weeks), before performing a high repetition high force (HRHF) task for 3 weeks (2 h/day, 3 days/week). HRHF rats were untreated, or treated in task weeks 2 and 3 with a monoclonal antibody that blocks CCN2 (FG-3019), or a control immunoglobulin G (IgG). Control rats were untreated or received FG-3019, IgG, or vehicle (saline) injections. Mean task reach rate and grasp force were higher in 3-week HRHF + FG-3019 rats, compared with untreated HRHF rats. Grip strength declined while forepaw mechanical sensitivity increased in untreated HRHF rats, compared with controls; changes improved by FG-3019 treatment. The HRHF task increased collagen in multiple tissues (flexor digitorum muscles, nerves, and forepaw dermis), which was reduced with FG-3019 treatment. FG-3019 treatment also reduced HRHF-induced increases in CCN2 and transforming growth factor ß in muscles. In tendons, FG-3019 reduced HRHF-induced increases in CCN2, epitendon thickening, and cell proliferation. Our findings indicate that CCN2 is critical to the progression of chronic overuse-induced multi-tissue fibrosis and functional declines. FG-3019 treatment may be a novel therapeutic strategy for overuse-induced musculoskeletal disorders. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2004-2018, 2019.

Network Analysis of Depression-Related Transcriptomic Profiles.

Major depressive disorder is a common debilitating disorder that is associated with increased morbidity and mortality. However, the molecular mechanism underlying depression remains largely unknown. The current study investigated the association of depression with blood gene expression using data from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Depression was measured by the geriatric depression scale, and the blood gene expression was measured by the Affymetrix Human Genome U219 Array. Linear regression was used to test the association between gene expression and depression, and the model was adjusted for age and sex. A total of 671 participants were included in our study (mean age 75 ± 8 years, 43.2% women). We found three genes were associated with depression, including COL1A2 (P = 8.9 × 10-8), RNF150 (P = 1.4 × 10-7) and CTGF (P = 8.3 × 10-7). An interaction network was built, and the pathway analysis indicated that many depression-related genes were involved in the neurotrophin signaling pathway (P = 2.1 × 10-7). Future studies are necessary to validate our findings and further investigate potential mechanism of depression.

Significance of CCN2 expression in bovine preimplantation development.

In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species-specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency-related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.

Clinical evidence and mechanisms of growth factors in idiopathic and diabetes-induced carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is an entrapment neuropathy caused by compression and irritation of the median nerve, which travels through the carpal tunnel in the wrist. Increased fibrosis is a hallmark of the development and pathology of CTS. Different growth factors have been demonstrated to play a potential role in the development of CTS. Studies have described an increase in the expression of growth factors, including Transforming Growth Factor (TGF-ß), Vascular Endothelial Growth Factor (VEGF) and interleukins (growth factors for immune and inflammatory cells) in SSCT (sub-synovial connective tissue) in CTS patients. Additionally, SSCT fibrosis is also marked by increased activation of canonical TGF-ß second messenger Smads, increased expression of downstream fibrotic mediators such as connective tissue growth factor (CTGF), increased production of collagen type I, II, III and IV, and decreased expression of matrix metalloproteinases. Anti-fibrotic such as anti-TGF treatment may prove beneficial in idiopathic patients, however, anti VEGF therapy can be successful in the diabetic CTS patients. The present review describes the clinical evidence stating the role of different growth factors in the development of fibrosis in idiopathic and diabetes induced CTS.

Connective tissue growth factor contributes to joint homeostasis and osteoarthritis severity by controlling the matrix sequestration and activation of latent TGFß.

OBJECTIVES: One mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue growth factor (CTGF) and here investigate its function and mechanism of action. METHODS: Recombinant CTGF (rCTGF) was used to stimulate human chondrocytes for microarray analysis. Endogenous CTGF was investigated by in vitro binding assays and confocal microscopy. Its release from cut cartilage (injury CM) was analysed by Western blot under reducing and non-reducing conditions. A postnatal, conditional CtgfcKO mouse was generated for cartilage injury experiments and to explore the course of osteoarthritis (OA) by destabilisation of the medial meniscus. siRNA knockdown was performed on isolated human chondrocytes. RESULTS: The biological responses of rCTGF were TGFß dependent. CTGF displaced latent TGFß from cartilage and both were released on cartilage injury. CTGF and latent TGFß migrated as a single high molecular weight band under non-reducing conditions, suggesting that they were in a covalent (disulfide) complex. This was confirmed by immunoprecipitation. Using CtgfcKO mice, CTGF was required for sequestration of latent TGFß in the matrix and activation of the latent complex at the cell surface through TGFßR3. In vivo deletion of CTGF increased the thickness of the articular cartilage and protected mice from OA. CONCLUSIONS: CTGF is a latent TGFß binding protein that controls the matrix sequestration and activation of TGFß in cartilage. Deletion of CTGF in vivo caused a paradoxical increase in Smad2 phosphorylation resulting in thicker cartilage that was protected from OA.

CTGF Contributes to the Development of Posterior Capsule Opacification: an in vitro and in vivo study.

Connective tissue growth factor (CTGF) is a crucial factor that plays a major role in the process of posterior capsule opacification (PCO). However, the effects of CTGF on the proliferation and migration of lens epithelial cells (LECs) and on the mechanism of the epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) in human lens epithelial cells (HLECs) as well as the effects of shRNA-mediated CTGF knockdown on the development of PCO in rats remain unclear. In the present study, we found that CTGF promoted EMT, proliferation, migration and the expression of p-ERK1/2 protein in HLECs but exerted little effect on the expression of p-p38 and p-JNK1/2 proteins. MEK inhibitor U0126 effectively restrained the CTGF-induced expression of α-smooth muscle actin (α-SMA), fibronectin (Fn) and type I collagen (COL-1) in HLECs. CTGF knockdown effectively postponed the onset of PCO in the rats and significantly reduced the expression of α-SMA in the capsule. In conclusion, CTGF contributed to the development of PCO presumably by promoting proliferation, migration of LECs, EMT specific protein expression and ECM synthesis in HLECs, which is dependent on ERK signalling. Furthermore, blocking CTGF effectively inhibited PCO in the rats and the EMT specific protein expression in the lens capsule.