Connective Tissue Growth Factor Research Articles

Distinct distribution of subplate neuron subtypes between the sensory cortices during the early postnatal period.

Subplate neurons (SpNs) are a heterogeneous neuronal population actively involved in early cortical circuit formation. In rodents, many SpNs survive and form layer 6b. The molecular heterogeneity of SpNs raises the question of whether different subpopulations of SpNs survive through the early postnatal period similarly and whether such diverse SpN populations in the auditory cortex(ACtx) share a common distribution pattern with other sensory systems. To address that, we investigated the expression pattern of multiple specific SpN markers in the ACtx, as well as in the visual (VCtx) and somatosensory (SCtx) cortices as controls, using complexin 3 (Cplx3) antibodies and different SpN-specific Cre-driver mice, such as connective tissue growth factor (CTGF), dopamine receptor D1 (Drd1a), and neurexophilin 4 (Nxph4). We focused on two early time windows in auditory development: (1) during the second postnatal week (PNW) before ear-canal opening and (2) during the third PNW after ear-canal opening. We compared the expression pattern of different SpN markers in ACtx with VCtx and SCtx. At both examined timepoints, Cplx3 and Nxph4 expressing SpNs form the largest and smallest population in the ACtx, respectively. Similar distribution patterns are observable in the VCtx and SCtx during the second PNW but not during the third PNW, for a higher proportion of Drd1a expressing SpNs is detected in the VCtx and CTGF expressing SpNs in the SCtx. This study suggests that different populations of SpNs might contribute differently to the development of individual sensory circuits.

Impact of Standard Versus Low Pneumoperitoneum Pressure on Peritoneal Environment in Laparoscopic Cholecystectomy. Randomized Clinical Trial.

High CO 2 pneumoperitoneum pressure during laparoscopy adversely affects the peritoneal environment. This study hypothesized that low pneumoperitoneum pressure may be linked to less peritoneal damage and possibly to better clinical outcomes. One hundred patients undergoing scheduled laparoscopic cholecystectomy were randomized 1:1 to low or to standard pneumoperitoneum pressure. Peritoneal biopsies were performed at baseline time and 1 hour after peritoneum insufflation in all patients. The primary outcome was peritoneal remodeling biomarkers and apoptotic index. Secondary outcomes included biomarker differences at the studied times and some clinical variables such as length of hospital stay, and quality and safety issues related to the procedure. Peritoneal IL6 after 1 hour of surgery was significantly higher in the standard than in the low-pressure group (4.26±1.34 vs. 3.24±1.21; P =0.001). On the contrary, levels of connective tissue growth factor and plasminogen activator inhibitor-I were higher in the low-pressure group (0.89±0.61 vs. 0.61±0.84; P =0.025, and 0.74±0.89 vs. 0.24±1.15; P =0.028, respectively). Regarding apoptotic index, similar levels were found in both groups and were 44.0±10.9 and 42.5±17.8 in low and standard pressure groups, respectively. None of the secondary outcomes showed differences between the 2 groups. Peritoneal inflammation after laparoscopic cholecystectomy is higher when surgery is performed under standard pressure. Adhesion formation seems to be less in this group. The majority of patients undergoing surgery under low pressure were operated under optimal workspace conditions, regardless of the surgeon's expertise.

The pathogenic role of succinate-SUCNR1: a critical function that induces renal fibrosis via M2 macrophage

BackgroundRenal fibrosis significantly contributes to the progressive loss of kidney function in chronic kidney disease (CKD), with alternatively activated M2 macrophages playing a crucial role in this progression. The serum succinate level is consistently elevated in individuals with diabetes and obesity, both of which are critical factors contributing to CKD. However, it remains unclear whether elevated succinate levels can mediate M2 polarization of macrophages and contribute to renal interstitial fibrosis.MethodsMale C57/BL6 mice were administered water supplemented with 4% succinate for 12 weeks to assess its impact on renal interstitial fibrosis. Additionally, the significance of macrophages was confirmed in vivo by using clodronate liposomes to deplete them. Furthermore, we employed RAW 264.7 and NRK-49F cells to investigate the underlying molecular mechanisms.ResultsSuccinate caused renal interstitial macrophage infiltration, activation of profibrotic M2 phenotype, upregulation of profibrotic factors, and interstitial fibrosis. Treatment of clodronate liposomes markedly depleted macrophages and prevented the succinate-induced increase in profibrotic factors and fibrosis. Mechanically, succinate promoted CTGF transcription via triggering SUCNR1-p-Akt/p-GSK3β/β-catenin signaling, which was inhibited by SUCNR1 siRNA. The knockdown of succinate receptor (SUCNR1) or pretreatment of anti-CTGF(connective tissue growth factor) antibody suppressed the stimulating effects of succinate on RAW 264.7 and NRK-49F cells.ConclusionsThe causative effects of succinate on renal interstitial fibrosis were mediated by the activation of profibrotic M2 macrophages. Succinate-SUCNR1 played a role in activating p-Akt/p-GSK3β/β-catenin, CTGF expression, and facilitating crosstalk between macrophages and fibroblasts. Our findings suggest a promising strategy to prevent the progression of metabolic CKD by promoting the excretion of succinate in urine and/or using selective antagonists for SUCNR1.

Effect of electroacupuncture on ventricular structure and function in rats with myocardial ischemia-reperfusion injury.

To observe the effect of electroacupuncture (EA) on changes of ventricular structure and function in rats with myocardial ischemia-reperfusion injury (MIRI), so as to explore its potential mechanisms underlying improvement of ventricular remodeling after MIRI. Forty male SD rats were randomly divided into 4 groups：sham operation group, model group, EA group and medication (sacubactril valsartan, LCZ696) group, with 10 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery and reperfusion. EA (2 Hz/100 Hz, 2 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once every other day for 21 d. Rats of the medication group received gavage of LCZ696 (60 mg·kg-1·d-1). After the intervention, echocardiography was used to detect the ejection fraction (EF) and fractional shortening (FS) of the left ventricle, and the contents of serum tumor necrosis factor-α(TNF-α), vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1) were assayed by enzyme-linked immunosorbent assay. The pathological changes of myocardial tissue were observed after HE staining. The Masson staining was used to evaluate the myocardial collagen deposition and myocardial fibrosis. The mRNA expression levels of collagen Ⅰ and Ⅲ and connective tissue growth factor (CTGF) in the myocardial tissue were detected by quantitative real-time PCR, and the expression levels of IL-1β and IL-18 were detected by Western blot. In contrast to the sham operation group, the EF and FS levels of the left ventricle were ob-viously decreased (P<0.001), while the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, the expression levels of IL-1β and IL-18 were significantly increased (P<0.001, P<0.000 1, P<0.05, P<0.01) in the model group. Compared with the model group, the EF and FS levels were remarkably increased (P<0.01), whereas the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, and the expression levels of IL-1β and IL-18 were significantly down-regulated (P<0.001, P<0.01, P<0.05) in both the medication and EA groups. No significant differences were found between the EA and medication groups in all the indexes mentioned above. EA can improve the left-ventricular fibrosis and function, delay or reverse ventricular remodeling in MIRI rats, which may be related to its functions in down-regulating myocardial inflammatory response and mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF.

Developmental morphogens direct human induced pluripotent stem cells toward an annulus fibrosus-like cell phenotype.

Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown. iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-β3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells. TGF-β3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-β3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-β3 and PDGF-BB for 14 days. These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies.

Aerobic exercise protects MI heart through miR-133a-3p downregulation of connective tissue growth factor.

To investigate the effect of aerobic exercise intervention to inhibit cardiomyocyte apoptosis and thus improve cardiac function in myocardial infarction (MI) mice by regulating CTGF expression through miR-133a-3p. Male C57/BL6 mice, 7-8 weeks old, were randomly divided into sham-operated group (S group), sham-operated +aerobic exercise group (SE group), myocardial infarction group (MI group) and MI + aerobic exercise group (ME group). The mice were anesthetized the day after training and cardiac function was assessed by cardiac echocardiography. Myocardial collagen volume fraction (CVF%) was analyzed by Masson staining. Myocardial CTGF, Bax and Bcl-2 were detected by Western blotting, and myocardial miR-133a-3p was measured by RT-qPCR. Compared with the S group, miR-133a-3p, Bcl-2 and EF were significantly decreased and CTGF, Bax, Bax/ Bcl-2, Caspase 3, Cleaved Caspase-3, LVIDd, LVIDs and CVF were significantly increased in the MI group. Compared with the MI group, miR-133a-3p, Bcl-2 and EF were significantly increased, cardiac function was significantly improved, and CTGF, Bax, Bax/ Bcl-2, Caspase 3, Cleaved Caspase-3, LVIDd, LVIDs and CVF were significantly decreased in ME group. The miR-133a-3p was significantly lower and CTGF was significantly higher in the H2O2 intervention group compared with the control group of H9C2 rat cardiomyocytes. miR-133a-3p was significantly higher and CTGF was significantly lower in the AICAR intervention group compared to the H2O2 intervention group. Compared with the control group of H9C2 rat cardiomyocytes, CTGF, Bax and Bax/Bcl-2 were significantly increased and Bcl-2 was significantly decreased in the miR-133a-3p inhibitor intervention group; CTGF, Bax and Bax/Bcl-2 were significantly decreased and Bcl-2 was significantly upregulated in the miR-133a-3p mimics intervention group. Aerobic exercise down-regulated CTGF expression in MI mouse myocardium through miR-133a-3p, thereby inhibiting cardiomyocyte apoptosis and improving cardiac function.

Sinapic acid modulates oxidative stress and metabolic disturbances to attenuate ovarian fibrosis in letrozole-induced polycystic ovary syndrome SD rats.

Sinapic acid (SA) is renowned for its many pharmacological activities as a polyphenolic compound. The cause of polycystic ovary syndrome (PCOS), a commonly encountered array of metabolic and hormonal abnormalities in females, has yet to be determined. The present experiment was performed to evaluate the antifibrotic properties of SA in rats with letrozole-induced PCOS-related ovarian fibrosis. SA treatment successfully mitigated the changes induced by letrozole in body weight (BW) (p < .01) and relative ovary weight (p < .05). Histological observation revealed that SA reduced the number of atretic and cystic follicles (AFs) and (CFs) (p < .01), as well as ovarian fibrosis, in PCOS rats. Additionally, SA treatment impacted the serum levels of sex hormones in PCOS rats. Luteinizing hormone (LH) and testosterone (T) levels were decreased (p < .01, p < .05), and follicle-stimulating hormone (FSH) levels were increased (p < .05). SA administration also decreased triglyceride (TG) (p < .01) and total cholesterol (TC) levels (p < .05) and increased high-density lipoprotein cholesterol (HDL-C) levels (p < .01), thereby alleviating letrozole-induced metabolic dysfunction in PCOS rats. Furthermore, SA treatment targeted insulin resistance (IR) and increased the messenger RNA (mRNA) levels of antioxidant enzymes in the ovaries of PCOS rats. Finally, SA treatment enhanced the activity of peroxisome proliferator-activated receptor-γ (PPAR-γ), reduced the activation of transforming growth factor-β1 (TGF-β1)/Smads, and decreased collagen I, α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF) levels in the ovaries of PCOS rats. These observations suggest that SA significantly ameliorates metabolic dysfunction and oxidative stress and ultimately reduces ovarian fibrosis in rats with letrozole-induced PCOS.

Connective tissue growth factor enhances TGF-β1-induced osteogenic differentiation via activation of p38 MAPK in mesenchymal stem cells

ObjectivesCellular differentiation is based on the effects of various growth factors. Transforming growth factor (TGF)-β1 plays a pivotal role in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the influence of connective tissue growth factor (CTGF), known to function synergistically with TGF-β1, on osteogenic differentiation in MSCs. MethodsUE7T-13 cells were treated with TGF-β1 and/or CTGF. Subsequently, protein levels of intracellular signaling pathway molecules were determined through western blot analysis. The mRNA expression levels of osteogenic differentiation markers were investigated using reverse transcription-quantitative polymerase chain reaction. Bone matrix mineralization was evaluated through alizarin red staining. ResultsCo-treatment with TGF-β1 and CTGF resulted in the suppression of TGF-β1-induced phosphorylation of extracellular signal-regulated kinase 1/2, an intracellular signaling pathway molecule in MSCs, while significantly enhancing the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In MSCs, co-treatment with CTGF and TGF-β1 led to increased expression levels of alkaline phosphatase and type I collagen, markers of osteogenic differentiation induced by TGF-β1. Osteopontin expression was observed only after TGF-β1 and CTGF co-treatment. Notably, bone sialoprotein and osteocalcin were significantly upregulated by treatment with CTGF alone. Furthermore, CTGF enhanced the TGF-β1-induced mineralization in MSCs, with complete suppression observed after treatment with a p38 MAPK inhibitor. ConclusionsCTGF enhances TGF-β1-induced osteogenic differentiation and subsequent mineralization in MSCs by predominantly activating the p38 MAPK-dependent pathway.

Research of miR-29a on TGF-β1/Smad3 pathway in pulmonary fibrosis induced by neodymium oxide

Objective: To exploring the regulatory effect of miR-29a on the transforming growth factor-β1 (TGF-β1) /Smad homolog 3 (Smad3) pathway during the process of rare earth neodymium oxide (Nd(2)O(3)) induced pulmonary fibrosis in mice. Methods: In March 2021, 72 SPF grade C57/BL6J male mice were selected and randomly divided into a control group, Nd(2)O(3) group, Nd(2)O(3)+miR-29a agomir group, and Nd(2)O(3)+NC agomir group, with 18 mice in each group. The Nd(2)O(3) group, Nd(2)O(3)+miR-29a agomir group, and Nd(2)O(3)+NC agomir group were treated with non exposed tracheal instillation, with a dust concentration of 250 mg/ml and a dust volume of 0.1 ml. The control group was given the same volume of physiological saline. After exposure to Nd(2)O(3), 0.1 ml (5 nmol) of miR-29a agomir was injected into the tail vein of mice in the Nd(2)O(3)+miR-29a agomir group every 3 days, while 0.1 ml of NC agomir was injected into the tail vein of mice in the Nd(2)O(3)+NC agomir group. On the 7 th, 14 th, and 28 th days after dust exposure, 6 mice were killed in each group, and the lung tissue of the mice was taken out. HE staining was used to observe the pathological status of the mouse lung tissue; ELISA method was used to detect the levels of TGF-β1 and connective tissue growth factor (CTGF) in lung tissue; Use qRT-PCR detection method to detect the expression level of TGF-β1 mRNA; Using immunofluorescence assay to detect the expression level of Smad3 in mouse lung tissue; Use bioinformatics websites such as TargetScan7 and miRDB to predict the target gene of miR-29a. When the metrological date were satisfied with normal distribution, Mean±SD was used for comparison between groups, t test was used for two indepent samples, and LSD method was used when the variance was homogeneity in pairwise comparison. Results: HE staining showed that the Nd(2)O(3) group of mice showed obvious infiltration of inflammatory cells and structural disorder of alveoli in the early stage of lung tissue. At 28 days, the collagen fibers in the mouse lung tissue increased and the lung tissue showed fibrotic honeycomb like changes. The degree of pulmonary fibrosis in the Nd(2)O(3)+miR-29a agomir group of mice was significantly reduced; The content of TGF-β1 and CTGF in the lung tissue of mice in the Nd(2)O(3)+miR-29a agomir group was lower than that in the Nd(2)O(3)+NC agomir group (P<0.05) ; The relative expression level of TGF-β1 in the lung tissue of mice in the Nd(2)O(3)+miR-29a agomir group was lower than that in the Nd(2)O(3)+NC agomir group (P<0.05) ; The expression level of Smad3 in the nucleus of the Nd(2)O(3)+miR-29a agomir group was lower than that of the Nd(2)O(3)+NC agomir group (P<0.05). The prediction results of bioinformatics websites have found 152 downstream target genes related to miR-29a, among which FBN1, MAP2K6, KPNB1, COL1A2, SNIP1, LAMC1, and SP1 genes may be related to the regulatory effect of miR-29a on TGF-β1/Smad3 signaling pathway. Conclusion: miR-29a may affect lung fibrosis induced by rare earth Nd(2)O(3) exposure in mice by regulating TGF-β1/Smad3 signaling pathway. Overexpression of miR-29a may inhibit TGF-β1/Smad3 signaling pathway and reduce the degree of pulmonary fibrosis in mice.

Impact of umbelliprenin-containing niosome nanoparticles on VEGF-A and CTGF genes expression in retinal pigment epithelium cells.

To investigate the impact of niosome nanoparticles carrying umbelliprenin (UMB), an anti-angiogenic and anti-inflammatory plant compound, on the expression of vascular endothelial growth factor (VEGF-A) and connective tissue growth factor (CTGF) genes in a human retinal pigment epithelium (RPE)-like retina-derived cell line. UMB-containing niosomes were created, optimized, and characterized. RPE-like cells were treated with free UMB and UMB-containing niosomes. The IC50 values of the treatments were determined using an MTT assay. Gene expression of VEGF-A and CTGF was evaluated using real-time polymerase chain reaction after RNA extraction and cDNA synthesis. Niosomes' characteristics, including drug entrapment efficiency, size, dispersion index, and zeta potential were assessed. Free UMB had an IC50 of 96.2 µg/mL, while UMB-containing niosomes had an IC50 of 25 µg/mL. Treatment with UMB-containing niosomes and free UMB resulted in a significant reduction in VEGF-A expression compared to control cells (P=0.001). Additionally, UMB-containing niosomes demonstrated a significant reduction in CTGF expression compared to control cells (P=0.05). However, there was no significant reduction in the expression of both genes in cells treated with free UMB. Both free UMB and niosome-encapsulated UMB inhibits VEGF-A and CTGF genes expression. However, the latter demonstrates significantly greater efficacy, potentially due to the lower UMB dosage and gradual delivery. These findings have implications for anti-angiogenesis therapeutic approaches targeting age-related macular degeneration.

Expression of the Pro-Fibrotic Marker Periostin in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by fibrotic tissue deposition in skeletal muscle. We assessed the role of periostin in fibrosis using mdx mice, an established DMD murine model, for which we conducted a thorough examination of periostin expression over a year. RNA and protein levels in diaphragm (DIA) muscles were assessed and complemented by a detailed histological analysis at 5 months of age. In dystrophic DIAs, periostin (Postn) mRNA expression significantly exceeded that seen in wildtype controls at all timepoints analysed, with the highest expression at 5 months of age (p < 0.05). We found Postn to be more consistently highly expressed at the earlier timepoints compared to established markers of fibrosis like transforming growth factor-beta 1 (Tgf-β1) and connective tissue growth factor (Ctgf). Immunohistochemistry confirmed a significantly higher periostin protein expression in 5-month-old mdx mice compared to age-matched healthy controls (p < 0.01), coinciding with a significant fibrotic area percentage (p < 0.0001). RT-qPCR also indicated an elevated expression of Tgf-β1, Col1α1 (collagen type 1 alpha 1) and Ctgf in mdx DIAs compared to wild type controls (p < 0.05) at 8- and 12-month timepoints. Accordingly, immunoblot quantification demonstrated elevated periostin (3, 5 and 8 months, p < 0.01) and Tgf-β1 (8 and 12 months, p < 0.001) proteins in the mdx muscle. These findings collectively suggest that periostin expression is a valuable marker of fibrosis in this relevant model of DMD. They also suggest periostin as a potential contributor to fibrosis development, with an early onset of expression, thereby offering the potential for timely therapeutic intervention and its use as a biomarker in muscular dystrophies.

A new lymphedema treatment using pyro-drive jet injection.

Lymphedema, resulting from impaired lymphatic drainage, causes inflammation, fibrosis and tissue damage leading to symptoms such as limb swelling and restricted mobility. Despite various treatments under exploration, no standard effective therapy exists. Here a novel technique using the pyro-drive jet injection (PJI) was used to create artificial clefts between collagen fibers, which facilitated the removal of excess interstitial fluid. The PJI was used to deliver a mixture of lactated Ringer's solution and air into the tail of animals with secondary skin edema. Edema levels were assessed using micro-CT scanning. Histopathological changes and neovascularization were evaluated on the injury-induced regenerative tissue. Regarding tissue remodeling, we focused on connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF)-C. PJI markedly diminished soft tissue volume in the experimental lymphedema animals compared to the non-injected counterparts. The PJI groups exhibited a significantly reduced proportion of inflammatory granulation tissue and an enhanced density of lymphatic vessels and α-smooth muscle actin (αSMA)-positive small vessels in the fibrous granulation tissue compared to the controls. In addition, PJI curtailed the prevalence of CTGF- and VEGF-C-positive cells in regenerative tissue. In a lymphedema animal model, PJI notably ameliorated interstitial edema, promoted lymphatic vessel growth, and bolstered αSMA-positive capillaries in fibrous granulation tissue. PJI's minimal tissue impact post-lymph node dissection indicates significant potential as an early, standard preventative measure. Easily applied in general clinics without requiring specialized training, it offers a cost-effective and highly versatile solution to the management of lymphedema.

Photocrosslinkable silk-based bioinks embedded with growth factor loaded microspheres towards cell-free 3D printing approach for biomimetic regeneration of meniscal tissue

Fibrocartilaginous meniscal tear regeneration is a major challenge due to the large avascular regions which suffer several injuries among all age-groups. Mostly, meniscus implants are focused on the development of multi-layered meniscus constructs and scaffolds that mimic the macro-architecture, functionality and mechanical strength. The native human menisci consist of vascular and avascular regions that contain different cellular phenotypes and are hence composed of a gradient of extracellular matrix (ECM) components. The biochemical composition of the inner, middle and outer regions of the knee menisci plays a significant role in the mechanical behavior and its overall functionality. We have developed a photo-polymerizing mulberry and non-mulberry silk-based biomaterial ink which is laden with growth factor loaded gelMA microspheres for the zone-specific regeneration of full thickness meniscus tears using three-dimensional (3D) fabrication. We have fabricated growth factor loaded gelMA microspheres and encapsulated them within the biomaterial ink for controlled release of Connective Tissue Growth Factor (CTGF) and Transforming Growth Factor-beta3 (TGF-β3) from the different zones of the meniscus constructs. The growth factor releasing constructs demonstrate the requisite mechanical resilience, degradability and release behaviour for regeneration of meniscal tissue. The developed growth factor loaded constructs demonstrate zone specific phenotypic differentiation of adipose derived stem cells and extracellular matrix deposition by the differentiated stem cells in vitro. The microsphere loaded constructs were found to be immuno-compatible as well. Therefore, these bioactive microsphere-laden 3D printed meniscus constructs would potentially promote regeneration and integration of meniscal tissue in vivo.

Serum level of full-length connective tissue growth factor reflects liver fibrosis stage in patients with Fontan-associated liver disease.

Chronic liver disease leads to liver fibrosis, and an accurate diagnosis of the fibrosis stage is crucial for medical management. Connective tissue growth factor (CTGF) is produced by endothelial cells and platelets and plays a central role in inducing fibrosis in various organs. In the present study, we tested the validity of measuring the serum levels of two types of CTGF to estimate the biopsy-confirmed liver fibrosis stage. We used two detection antibodies targeting the N- and C-terminal of CTGF to measure the serum levels of two forms of CTGF consisting of its full length and its N-terminal fragment. We analyzed the level of CTGF (via enzyme-linked immunosorbent assay) and the liver fibrosis stage in 38 patients with Fontan-associated liver disease (FALD) (26 cases of which were diagnosed pathologically). Correlations were determined by multivariate analysis and the area under the receiver operating characteristic curve. The 65 patients with nonalcoholic fatty liver disease (NAFLD) were included as a disease control group for examination. Full-length CTGF was significantly inversely correlated with liver fibrosis in patients with FALD. Although the platelet count was also associated with the liver fibrosis stage, full-length CTGF was more closely correlated with the fibrosis stage. Furthermore, the level of full-length CTGF was inversely associated with high central venous pressure. Conversely, the serum level of CTGF was not correlated with the fibrosis stage in NAFLD. The serum level of full-length CTGF may be useful for estimating the liver fibrosis stage in patients with FALD.

Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts

BackgroundCancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that play an important role in cancer progression. Although the mechanism by which CAFs promote tumorigenesis has been well investigated, the underlying mechanism of CAFs activation by neighboring cancer cells remains elusive. In this study, we aim to investigate the signaling pathways involved in CAFs activation by gastric cancer cells (GC) and to provide insights into the therapeutic targeting of CAFs for overcoming GC.MethodsAlteration of receptor tyrosine kinase (RTK) activity in CAFs was analyzed using phospho-RTK array. The expression of CAFs effector genes was determined by RT-qPCR or ELISA. The migration and invasion of GC cells co-cultured with CAFs were examined by transwell migration/invasion assay.ResultsWe found that conditioned media (CM) from GC cells could activate multiple receptor tyrosine kinase signaling pathways, including ERK, AKT, and STAT3. Phospho-RTK array analysis showed that CM from GC cells activated PDGFR tyrosine phosphorylation, but only AKT activation was PDGFR-dependent. Furthermore, we found that connective tissue growth factor (CTGF), a member of the CCN family, was the most pronouncedly induced CAFs effector gene by GC cells. Knockdown of CTGF impaired the ability of CAFs to promote GC cell migration and invasion. Although the PDGFR-AKT pathway was pronouncedly activated in CAFs stimulated by GC cells, its pharmacological inhibition affected neither CTGF induction nor CAFs-induced GC cell migration. Unexpectedly, the knockdown of SRC and SRC-family kinase inhibitors, dasatinib and saracatinib, significantly impaired CTGF induction in activated CAFs and the migration of GC cells co-cultured with CAFs. SRC inhibitors restored the reduced expression of epithelial markers, E-cadherin and Zonula Occludens-1 (ZO-1), in GC cells co-cultured with CAFs, as well as CAFs-induced aggregate formation in a 3D tumor spheroid model.ConclusionsThis study provides a characterization of the signaling pathways and effector genes involved in CAFs activation, and strategies that could effectively inhibit it in the context of GC.DC5ErEG73tsyg3F8WfE1xiVideo

YAP1 expression in colorectal cancer confers the aggressive phenotypes via its target genes

ABSTRACT Yes-associated protein1 (YAP1), a downstream effector of the Hippo pathway, is over-expressed in several types of malignancies. We analyzed retrospectively the TCGA database using 447 colorectal cancer (CRC) samples to determine the correlation between YAP1 expression level and CRC patient prognosis. YAP1-enforced expressed CRC cell lines were constructed using the lentivirus particles containing a YAP1 insert. YAP1 was highly expressed in CRC cancerous tissues and is associated with distant metastasis of CRC patients. Kaplan – Meier analysis indicated that CRC patients with a higher YAP1 expression group (n = 104) had worse disease-free survival (DFS) and overall survival (OS) than lower YAP1 expression group (n = 343) (p = 0.008 and p = 0.022). Univariate and multivariate analysis indicated that the elevated YAP1 expression predicted the aggressive phenotype and was an independent indicator for OS and DFS of CRC patients. YAP1 over-expression in CRC cells enhanced their migration and invasion significantly which can be reversed by AXL, CTGF, or CYR61 interference. The study suggested that YAP1 affected the prognosis of CRC patients and controlled the abilities of invasion and migration of CRC cells via its target genes AXL, CTGF, and CYR61.

Claudin-2 Mediates the Proximal Tubular Epithelial Cell-Fibroblast Crosstalk via Paracrine CTGF.

Proximal tubular epithelial cell (PTEC) is vulnerable to injury in diabetic kidney disease (DKD) due to high energy expenditure. The injured PTECs-derived profibrotic factors are thought to be driving forces in tubulointerstitial fibrosis (TIF) as they activate surrounding fibroblasts. However, the mechanisms remain unclear. The diabetes with uninephrectomy (DKD) rats were used to evaluated renal histological changes and the expression of Claudin-2 by immunofluorescence staining. Then, Claudin-2 expression in PTECs were modulated and subsequently determined the proliferation and activation of fibroblasts by building a transwell co-culture system in normal glucose (NG)or high glucose (HG) condition. Decreased expression of Claudin-2 in PTECs accompanied by tight junction disruption and increased interstitial fibrosis, were detected in DKD rats. In vitro, downregulated Claudin-2 in PTECs promoted proliferation and activation of fibroblasts, which coincided with elevated expression of profibrotic connective tissue growth factor (CTGF) in PTECs. Silenced CTGF inhibited the profibrotic of PTECs via Claudin-2 inhibition. Fibroblasts co-cultured with PTECs transitioned more to myofibroblasts and generated extracellular matrix (ECM) significantly in response to high glucose (HG) stimulation whereas overexpression of Claudin-2 in PTECs reversed the above results. Upregulating CTGF disrupted the beneficial anti-fibrosis effects obtained by overexpression of Claudin-2 in HG condition. Our study suggested that Claudin-2 in PTECs, a key mediator of paracellular cation and water transport, promotes the activation and proliferation of surrounding fibroblasts significantly via CTGF in a paracrine manner.

Connective tissue growth factor among elderly patients with type 2 diabetes: Correlation with diabetic nephropathy

Connective tissue growth factor among elderly patients with type 2 diabetes: Correlation with diabetic nephropathy

A new procedure to induce aortic aneurysms in mice.

Aortic aneurysms (AAs) are a major public health challenge, featured by a progressive impairs in aortic wall integrity that drives to aortic dilation and, in end stage, to its rupture. Despite important advances in the surgical treatment of aortic aneurysms, there is currently no pharmacological intervention that prevents their development, reduces their expansion, or avoids their rupture. In addition to classic risk factors such age or gender, several heritable connective tissue disorders have been associated with AA developing, highlighting the role of extracellular matrix (ECM) genes alterations in the developing of AA. In this sense, we have recently demonstrated that global deletion of the cellular communicating network factor 2 (CCN2), previously known as connective tissue growth factor (CTGF) due to its role in the extracellular matrix formation, predisposes to early and lethal AAs development after Angiotensin II (Ang II) infusion in mice. Here, we detail the protocol to induce and detect AAs generation in inducible global CCN2 knockout mice after Ang II infusion which allow the characterization of CCN role in AA development and may help to the development of pharmacological target for AA treatment.

Fluvoxamine supresses transforming growth factor beta‐2‐induced fibrotic changes in trabecular meshwork cells

Aims/Purpose: Primary open‐angle glaucoma is the leading cause of blindness worldwide, but no definitive treatment is available. Fibrotic‐like changes of trabecular meshwork (TM) results in increased stiffness and impaired outflow leading to increased IOP. Transforming growth factor‐β2 (TGF‐β2), a main driver of eye fibrosis, is increased in the aqueous humour of glaucoma patients. Recently, we proved that the specific sigma‐1 receptor agonist fluvoxamine (FLU) is antifibrotic in the kidney but its role in the TM is unknown. Therefore, we investigated the effects of FLU on the TGF‐β2‐induced fibrotic response in TM cells.Methods: Fibrosis of the AS was induced in C57BL/6J mice by intracameral TGF‐β2 injection. AS were collected and F‐actin was visualized with Phalloidin and detected with confocal microscopy. The effect of FLU on TGF‐β2‐induced cell proliferation, cytoskeletal rearrangement, and fibrosis‐related proteins was investigated by MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide) assay, Phalloidin staining and Western blotting, respectively.Results: TGF‐β2 injection resulted in F‐actin enhancement in vivo, indicating fibrotic‐like changes. In vitro, TGF‐β2 induced cytoskeletal rearrangement with increased F‐actin‐ bundles and clumps formation (p &lt; 0.001) which were reduced by FLU (p &lt; 0.001). Furthermore, FLU also inhibited cell proliferation (p &lt; 0.05) in TGF‐β2‐treated cells. The profibrotic protein levels of connective tissue growth factor, fibronectin and collagen type IV were elevated by TGF‐β2 treatment (p &lt; 0.001), while FLU prevented the elevation of these elements (p &lt; 0.01; p &lt; 0.001). FLU also hindered the TGF‐β2 caused decrease of Matrix Metalloproteinase‐2 (p &lt; 0.05) thus facilitating ECM degradation.Conclusions: FLU may reduce the fibrotic response of trabecular meshwork and could be a potential candidate for the treatment of fibrosis‐induced ocular hypertension.Grants: OTKA‐ K135398, LP2021‐3/2021, TKP2021‐EGA‐24, Stipendium Hungaricum.