Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Modified climbing fiber/Purkinje cell synaptic connectivity in the cerebellum of the neonatal phencyclidine model of schizophrenia.

Environmental perturbations during the first years of life are a major factor in psychiatric diseases. Phencyclidine (PCP), a drug of abuse, has psychomimetic effects, and neonatal subchronic administration of PCP in rodents leads to long-term behavioral changes relevant for schizophrenia. The cerebellum is increasingly recognized for its role in diverse cognitive functions. However, little is known about potential cerebellar changes in models of schizophrenia. Here, we analyzed the characteristics of the cerebellum in the neonatal subchronic PCP model. We found that, while the global cerebellar cytoarchitecture and Purkinje cell spontaneous spiking properties are unchanged, climbing fiber/Purkinje cell synaptic connectivity is increased in juvenile mice. Neonatal subchronic administration of PCP is accompanied by increased cFos expression, a marker of neuronal activity, and transient modification of the neuronal surfaceome in the cerebellum. The largest change observed is the overexpression of Ctgf, a gene previously suggested as a biomarker for schizophrenia. This neonatal increase in Ctgf can be reproduced by increasing neuronal activity in the cerebellum during the second postnatal week using chemogenetics. However, it does not lead to increased climbing fiber/Purkinje cell connectivity in juvenile mice, showing the complexity of PCP action. Overall, our study shows that administration of the drug of abuse PCP during the developmental period of intense cerebellar synaptogenesis and circuit remodeling has long-term and specific effects on Purkinje cell connectivity and warrants the search for this type of synaptic changes in psychiatric diseases.

Drugs targeting CTGF in the treatment of pulmonary fibrosis.

Pulmonary fibrosis represents the final alteration seen in a wide variety of lung disorders characterized by increased fibroblast activity and the accumulation of substantial amounts of extracellular matrix, along with inflammatory damage and the breakdown of tissue architecture. This condition is marked by a significant mortality rate and a lack of effective treatments. The depositing of an excessive quantity of extracellular matrix protein follows the damage to lung capillaries and alveolar epithelial cells, leading to pulmonary fibrosis and irreversible damage to lung function. It has been proposed that the connective tissue growth factor (CTGF) plays a critical role in the advancement of pulmonary fibrosis by enhancing the accumulation of the extracellular matrix and exacerbating fibrosis. In this context, the significance of CTGF in pulmonary fibrosis is examined, and a summary of the development of drugs targeting CTGF for the treatment of pulmonary fibrosis is provided.

CD248-expressing cancer-associated fibroblasts induce non-small cell lung cancer metastasis via Hippo pathway-mediated extracellular matrix stiffness.

Metastasis is a crucial stage in tumour progression, and cancer-associated fibroblasts (CAFs) support metastasis through their participation in extracellular matrix (ECM) stiffness. CD248 is a possible biomarker for non-small cell lung cancer (NSCLC)-derived CAFs, but its role in mediating ECM stiffness to promote NSCLC metastasis is unknown. We investigated the significance of CD248+ CAFs in activating the Hippo axis and promoting connective tissue growth factor (CTGF) expression, which affects the stromal collagen I environment and improves ECM stiffness, thereby facilitating NSCLC metastasis. In this study, we found that higher levels of CD248 in CAFs induced the formation of collagen I, which in turn increased extracellular matrix stiffness, thereby enabling NSCLC cell infiltration and migration. Hippo axis activation by CD248+ CAFs induces CTGF expression, which facilitates the formation of the collagen I milieu in the stromal matrix. In a tumour lung metastasis model utilizing fibroblast-specific CD248 gene knockout mice, CD248 gene knockout mice showed a significantly reduced ability to develop tumour lung metastasis compared to that of WT mice. Our findings demonstrate that CD248+ CAFs activate the Hippo pathway, thereby inducing CTGF expression, which in turn facilitates the collagen I milieu of the stromal matrix, which promotes NSCLC metastasis.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Mechanical stress-induced connective tissue growth factor plays a critical role in intestinal fibrosis in Crohn's-like colitis.

Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of profibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMCs). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends toward normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.NEW & NOTEWORTHY We found that CTGF expression increased significantly not only in the inflammation site but in the distended segment proximal to inflammation in a rodent model of CD-like colitis. Release of mechanical distention prevented CTGF expression in CD rats, whereas direct stretch induced CTGF expression. Treatment with anti-CTGF antibody reduced fibrosis and collagen contents in CD rats. Thus, mechanical stress, via upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.

Effect of antibody-mediated connective tissue growth factor neutralization on lung edema in ventilator-induced lung injury in rats.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by alveolar edema that can progress to septal fibrosis. Mechanical ventilation can augment lung injury, termed ventilator-induced lung injury (VILI). Connective tissue growth factor (CTGF), a mediator of fibrosis, is increased in ARDS patients. Blocking CTGF inhibits fibrosis and possibly vascular leakage. This study investigated whether neutralizing CTGF reduces pulmonary edema in VILI. METHODS: Following LPS administration, rats were mechanically ventilated for 6 h with low (6 mL/kg; low VT) or moderate (10 mL/kg; mod VT) tidal volume and treated with a neutralizing CTGF antibody (FG-3154) or placebo lgG (vehicle). Control rats without LPS were ventilated for 6 h with low VT. Lung wet-to-dry weight ratio, FITC-labeled dextran permeability, histopathology, and soluble RAGE were determined. RESULTS: VILI was characterized by reduced PaO2/FiO2 ratio (low VT: 540 [381-661] vs. control: 693 [620-754], p < 0.05), increased wet-to-dry weight ratio (low VT: 4.8 [4.6-4.9] vs. control: 4.5 [4.4-4.6], p < 0.05), pneumonia (low VT: 30 [0-58] vs. control: 0 [0-0]%, p < 0.05) and interstitial inflammation (low VT: 2 [1-3] vs. control: 1 [0-1], p < 0.05). FG-3154 did not affect wet-to-dry weight ratio (mod VT + FG-3154: 4.8 [4.7-5.0] vs. mod VT + vehicle: 4.8 [4.8-5.0], p > 0.99), extravasated dextrans (mod VT + FG-3154: 0.06 [0.04-0.09] vs. mod VT + vehicle: 0.04 [0.03-0.09] µg/mg tissue, p > 0.99), sRAGE (mod VT + FG-3154: 1865 [1628-2252] vs. mod VT + vehicle: 1885 [1695-2159] pg/mL, p > 0.99) or histopathology. CONCLUSIONS: 'Double hit' VILI was characterized by inflammation, impaired oxygenation, pulmonary edema and histopathological lung injury. Blocking CTGF does not improve oxygenation nor reduce pulmonary edema in rats with VILI.

Ccn2a is an injury-induced matricellular factor that promotes cardiac regeneration in zebrafish.

The ability of zebrafish to heal their heart after injury makes them an attractive model for investigating the mechanisms governing the regenerative process. In this study, we show that the gene cellular communication network factor 2a (ccn2a), previously known as ctgfa, is induced in endocardial cells in the injured tissue and regulates CM proliferation and repopulation of the damaged tissue. We find that, whereas in wild-type animals, CMs track along the newly formed blood vessels that revascularize the injured tissue, in ccn2a mutants CM proliferation and repopulation are disrupted, despite apparently unaffected revascularization. In addition, we find that ccn2a overexpression enhances CM proliferation and improves the resolution of transient collagen deposition. Through loss- and gain-of-function as well as pharmacological approaches, we provide evidence that Ccn2a is necessary for and promotes heart regeneration by enhancing the expression of pro-regenerative extracellular matrix genes, and by inhibiting the chemokine receptor gene cxcr3.1 through a mechanism involving Tgfß/pSmad3 signaling. Thus, Ccn2a positively modulates the innate regenerative response of the adult zebrafish heart.

CTGF regulates mineralization in human mature chondrocyte by controlling Pit-1 and modulating ANK via the BMP/Smad signalling.

OBJECTIVE: Connective tissue growth factor (CTGF) exhibits potent proliferative, differentiated, and mineralizing effects, and is believed to be contribute to cartilage mineralization in Osteoarthritis (OA). However, the underlying mechanism of chondrocyte mineralization induced by CTGF remains obscure. As a key regulator of mineral responses, type III phosphate transporter 1 (Pit-1) has been associated with the pathogenesis of articular mineralization. Therefore, the primary objective of this study was to investigate whether CTGF influences the development of mature chondrocyte mineralization and the underlying mechanisms governing such mineralization. METHODS: The effect of Connective tissue growth factor (CTGF) on human C-28/I2 chondrocytes were investigated. The chondrocytes were treated with CTGF or related inhibitors, and transfected with Overexpression and siRNA transfection of Type III Phosphate Transporter 1(Pit-1). Subsequently, the cells were subjected to Alizarin red S staining, PiPer Phosphate Assay Kit, Alkaline Phosphatase Diethanolamine Activity Kit, ELISA, RT-PCR or Western blot analysis. RESULTS: Stimulation with Connective tissue growth factor (CTGF) significantly upregulated the expression of the Type III Phosphate Transporter 1(Pit-1) and mineralization levels in chondrocytes through activation of α5ß1 integrin and BMP/Samd1/5/8 signaling pathways. Furthermore, treatment with overexpressed Pit-1 markedly increased the expression of Multipass Transmembrane Ankylosis (ANK) transporter in the cells. The inhibitory effect of CTGF receptor blockade using α5ß1 Integrin blocking antibody was demonstrated by significantly suppressed the expression of Pit-1 and ANK transporter, as well as chondrocyte mineralization. CONCLUSIONS: Our data indicate that Connective tissue growth factor (CTGF) plays a critical role inchondrocyte mineralization, which is dependent on the expression of the Type III Phosphate Transporter 1(Pit-1) and Multipass Transmembrane Ankylosis (ANK) transporter. Consequently, inhibition of CTGF activity may represent a novel therapeutic approach for the management of Osteoarthritis (OA).

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

BACKGROUND: Cancer-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. METHODS: Alteration 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. RESULTS: We 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. CONCLUSIONS: This 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. Video Abstract.

Endothelial mesenchymal transition promotes pannus formation in ankylosing spondylitis by activation of TGF-ß/SMAD signalling pathway.

OBJECTIVES: To explore the role of endothelial-mesenchymal transition (EndMT) mediated by the TGF-ß/SMAD signalling pathway in the pathogenesis of ankylosing spondylitis (AS). METHODS: Serum levels of TGF-ß1 were measured by enzyme-linked immunosorbent assay (ELISA) in 48 patients with AS and 15 healthy subjects. The expression levels of TGF-ß1, SMAD7, CTGF, CD34 and EndMT-related markers (α-SMA, vimentin, FSP-1, VE-cadherin) in the sacroiliac joint (SIJ) of three AS patients were detected by immunohistochemistry, and three non-spondyloarthritis (SpA) autopsy samples were used as controls. RESULTS: Serum TGF-ß1 level of AS patients was significantly higher than that of healthy controls (22971 ± 7667 pg/ml vs. 14837±4653 pg/ml, p<0.01). Compared with the non-SpA control group, the microvascular density (MVD) at the pannus formation site of SIJ in AS patients was significantly increased, accompanied by respectively increased expressions of TGF-ß1, CTGF, α-SMA, vimentin, and FSP-1 (all p<0.05), whereas respectively decreased expressions of VE-cadherin and SMAD7 (p<0.01). The expression level of FSP-1 was positively correlated with levels of TGF-ß1 and MVD, and negatively correlated with SMAD7. CONCLUSIONS: Our findings show that EndMT is involved in the promotion of pannus formation by TGF-ß/SMAD signalling pathway activation in AS.

TGF-ß/Smad signaling pathway in fatty liver disease: a case-control study.

BACKGROUND: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-ß signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-ß signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups. METHODS AND RESULTS: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-ß1, TGF-ß3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-ß1 and TGF-ß3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001). CONCLUSIONS: TGF-ß signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-ß1 gene expression and its downstream signaling proteins more than TGF-ß3.

Exogenous S1P via S1P receptor 2 induces CTGF expression through Src-RhoA-ROCK-YAP pathway in hepatic stellate cells.

BACKGROUND: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial. METHODS: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation. RESULTS: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor. CONCLUSIONS: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.

Linagliptin ameliorates tacrolimus-induced renal injury: role of Nrf2/HO-1 and HIF-1α/CTGF/PAI-1.

BACKGROUND: Tacrolimus (TAC) is a frequently used immunosuppressive medication in organ transplantation. However, its nephrotoxic impact limits its long-term usage. This study aims to investigate the effect of linagliptin (Lina) on TAC-induced renal injury and its underlying mechanisms. METHODS AND RESULTS: Thirty-two Sprague Dawley rats were treated with TAC (1.5 mg/kg/day, subcutaneously) and/or Lina (5 mg/kg/day, orally) for 4 weeks. Histological examination was conducted, and serum and urinary biomarkers were measured to assess kidney function and integrity. Furthermore, ELISA, Western blot analysis and immunohistochemical assay were employed to determine signaling molecules of oxidative stress, profibrogenic, hypoxic, and apoptotic proteins. Tacrolimus caused renal dysfunction and histological deterioration evidenced by increased serum creatinine, blood urea nitrogen (BUN), urinary cystatin C, and decreased serum albumin as well as elevated tubular injury and interstitial fibrosis scores. Additionally, TAC significantly increased the expression of collagen type-1, alpha-smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor-beta1 (TGF-ß1) renal content. Moreover, TAC decreased the expression of nuclear factor erythroid-2-related factor2 (Nrf2), heme oxygenase 1 (HO-1), and mitochondrial superoxide dismutase (SOD2). In addition, TAC increased protein expression of hypoxia-inducible factor1-alpha (HIF-1α), connective tissue growth factor (CTGF), inducible nitric oxide synthase (iNOS), 8-hydroxy-2-deoxyguanosine (8-OHdG), as well as nitric oxide (NO), 4-hydroxynonenal, caspase-3 and Bax renal contents. Furthermore, TAC decreased Bcl-2 renal contents. The Lina administration markedly attenuated these alterations. CONCLUSION: Lina ameliorated TAC-induced kidney injury through modulation of oxidative stress, hypoxia, and apoptosis related proteins.

Down-regulation of CYTL1 attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting M2 macrophage polarization via the TGF-ß/CCN2 axis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor ß (TGF-ß)/connective tissue growth factor (CCN2) axis and inhibition of TGF-ß pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-ß/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.

Application of color doppler ultrasound and US shear wave elastography with connective tissue growth factor in the risk assessment of papillary thyroid carcinoma.

BACKGROUND: This study aims to investigate the role of shear wave elastography (SWE) and connective tissue growth factor (CTGF) in the assessment of papillary thyroid carcinoma (PTC) prognosis. METHODS: CTGF expression was detected with immunohistochemistry. Clinical and pathological data were collected. Parameters of conventional ultrasound combined with SWE were also collected. The relationship among CTGF expression, ultrasound indicators, the elastic modulus and the clinicopathological parameters were analyzed. RESULTS: Univariate analysis showed that patients with high risk of PTC were characterized with male, Uygur ethnicity, increased expression of CTGF, convex lesions, calcified, incomplete capsule, intranodular blood flow, rear echo attenuation, cervical lymph node metastasis, lesions larger than 1 cm, psammoma bodies, advanced clinical stage, increased TSH and high value in the shear modulus (P < 0.05). Multivariate analysis demonstrated that the risk factors of high expression of CTGF according to contribution size order were irregular shape, aspect ratio ≥ 1, and increased TSH. The logistic regression model equation was Logit (P) = 1.153 + 1.055 × 1 + 0.926 × 2 + 1.190 × 3 and the Area Under Curve value of the logistic regression was calculated to be 0.850, with a 95% confidence interval of 0.817 to 0.883. CONCLUSION: SWE and CTGF are of great value in the risk assessment of PTC. The degree of fibrosis of PTC is closely related to the prognosis. The hardness of PTC lesions and the expression level of CTGF are correlated with the main indexes of conventional ultrasound differentiating benign or malignant nodules. Irregular shape, aspect ratio ≥ 1, and increased TSH are independent factors of CTGF.

Neutralization of CX3CL1 Attenuates TGF-ß-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis.

BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-ß. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-ß-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). METHODS: CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-ß-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. RESULTS: An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-ß-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-ß-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-ß-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-ß-treated NHLFs. TP213 alleviated TGF-ß-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-ß-induced p65 and α-SMA expression in NHLFs. CONCLUSIONS: These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

Antiproliferative effect of low-level laser/ photobiomodulation on gingival fibroblasts derived from calcium channel blocker-induced gingival overgrowth.

The aim of this study was to evaluate the antiproliferative properties of low-level laser therapy (LLLT) on gingival fibroblasts obtained from calcium channel blocker-induced gingival overgrowth (GO). Gingival fibroblasts of patients with GO were compared to healthy gingival fibroblasts (H). Both cells were exposed to LLLT (685 nm wavelength, 25mW power, diode laser) and compared to those not treated with LLLT. Cell proliferation and viability were measured with MTT assay at baseline and after 24 and 72 h. TGF-ß1, CTGF, and collagen Type 1 levels were evaluated with Enzyme-Linked Immunosorbent Assay (ELISA). LLLT significantly decreased the proliferation of GO fibroblasts (p < 0.05) while leading to a significantly higher proliferation in H fibroblasts compared to the untreated cells (p < 0.05). GO cells showed significantly higher CTGF, TGF-ß, and collagen Type 1 expression than the H cells (p < 0.05). LLLT significantly reduced CTGF levels in GO cells compared to the control group (p < 0.05). In H cells, CTGF and TGF-ß levels were also significantly decreased in response to LLLT compared to the control group (p < 0.05). While LLLT significantly reduced collagen expression in the H group (p < 0.05), it did not significantly impact the GO cells. LLLT significantly reduced the synthesis of the growth factors and collagen in both groups with an antiproliferative effect on the gingival fibroblasts from calcium channel blocker-induced GO, suggesting that it can offer a therapeutic approach in the clinical management of drug-induced GO, reversing the fibrotic changes.

Lipid nephrotoxicity mediated by HIF-1α activation accelerates tubular injury in diabetic nephropathy.

This study is intended to explore the effect of hypoxia-inducible factor-1α (HIF-1α) activation on lipid accumulation in the diabetic kidney. A type 1 diabetic rat model was established by STZ intraperitoneal injection. Cobalt chloride (CoCl2) and YC-1 were used as the HIF-1α activator and antagonist, respectively. CoCl2 treatment significantly increased HIF-1α expression, accelerated lipid deposition, and accelerated tubular injury in diabetic kidneys. In vitro, CoCl2 effectively stabilized HIF-1α and increased its transportation from the cytoplasm to the nucleus, which was accompanied by significantly increased lipid accumulation in HK-2 cells. Furthermore, results obtained in vivo showed that HIF-1α protein expression in the renal tubules of diabetic rats was significantly downregulated by YC-1 treatment. Meanwhile, lipid accumulation in the tubules of the DM + YC-1 group was markedly decreased in comparison to the DM + DMSO group. Accordingly, PAS staining revealed that the pathological injury caused to the tubular epithelial cells was alleviated by YC-1 treatment. Furthermore, the blood glucose level, urine albumin creatinine ratio, and NAG creatinine ratio in the DM + YC-1 group were significantly decreased compared to the DM + DMSO group. Moreover, the protein expression levels of transforming growth factor ß1 (TGF-ß1) and connective tissue growth factor (CTGF) in diabetic kidneys were decreased by YC-1 treatment. Our findings demonstrate that the activation of HIF-1α contributed to interstitial injury in a rat model of diabetic nephropathy and that the underlying mechanism involved the induction of lipid accumulation.