Connective Tissue Growth Factor Research Articles

The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions.

To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium. During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

Pamrevlumab for Idiopathic Pulmonary Fibrosis: The ZEPHYRUS-1 Randomized Clinical Trial.

Current treatments for idiopathic pulmonary fibrosis slow the rate of lung function decline, but may be associated with adverse events that affect medication adherence. In phase 2 trials, pamrevlumab (a fully human monoclonal antibody that binds to and inhibits connective tissue growth factor activity) attenuated the progression of idiopathic pulmonary fibrosis without substantial adverse events. To assess the efficacy and safety of pamrevlumab for patients with idiopathic pulmonary fibrosis. Phase 3 randomized clinical trial including 356 patients aged 40 to 85 years with idiopathic pulmonary fibrosis who were not receiving antifibrotic treatment with nintedanib or pirfenidone at enrollment. Patients were recruited from 117 sites in 9 countries between July 18, 2019, and July 29, 2022; the last follow-up encounter occurred on August 28, 2023. Pamrevlumab (30 mg/kg administered intravenously every 3 weeks; n = 181) or placebo (n = 175) for 48 weeks. The primary outcome was absolute change in forced vital capacity (FVC) from baseline to week 48. There were 5 secondary outcomes (including time to disease progression, which was defined as a decline of ≥10% in predicted FVC or death). The exploratory outcomes included patient-reported symptoms. Adverse events were reported. Among 356 patients (mean age, 70.5 years; 258 [72.5%] were men; 221 [62.1%] were White), 277 (77.8%) completed the trial. There was no significant between-group difference for absolute change in FVC from baseline to week 48 (least-squares mean, -260 mL [95% CI, -350 to -170 mL] in the pamrevlumab group vs -330 mL [95% CI, -430 to -230 mL] in the placebo group; mean between-group difference, 70 mL [95% CI, -60 to 190 mL], P = .29). There were no significant between-group differences in any of the secondary outcomes or in the patient-reported outcomes. In the pamrevlumab group, there were 160 patients (88.4%) with treatment-related adverse events and 51 patients (28.2%) with serious adverse events vs 151 (86.3%) and 60 (34.3%), respectively, in the placebo group. During the study, 23 patients died in each group (12.7% in the pamrevlumab group vs 13.1% in the placebo group). Among patients with idiopathic pulmonary fibrosis treated with pamrevlumab or placebo, there was no statistically significant between-group difference for the primary outcome of absolute change in FVC from baseline to week 48. ClinicalTrials.gov Identifier: NCT03955146.

Development of alginate and alginate sulfate/polycaprolactone nanoparticles for growth factor delivery in wound healing therapy

Connective tissue growth factor (CTGF) holds great promise for enhancing the wound healing process; however, its clinical application is hindered by its low stability and the challenge of maintaining its effective concentration at the wound site. Herein, we developed novel double-emulsion alginate (Alg) and heparin-mimetic alginate sulfate (AlgSulf)/polycaprolactone (PCL) nanoparticles (NPs) for controlled CTGF delivery to promote accelerated wound healing. The NPs’ physicochemical properties, cytocompatibility, and wound healing activity were assessed on immortalized human keratinocytes (HaCaT), primary human dermal fibroblasts (HDF), and a murine cutaneous wound model. The synthesized NPs had a minimum hydrodynamic size of 200.25 nm. Treatment of HaCaT and HDF cells with Alg and AlgSulf2.0/PCL NPs did not show any toxicity when used at concentrations <50 µg/mL for up to 72 h. Moreover, the NPs’ size was not affected by elevated temperatures, acidic pH, or the presence of a protein-rich medium. The NPs have slow lysozyme-mediated degradation implying that they have an extended tissue retention time. Furthermore, we found that treatment of HaCaT and HDF cells with CTGF-loaded Alg and AlgSulf2.0/PCL NPs, respectively, induced rapid cell migration (76.12% and 79.49%, P<0.05). Finally, in vivo studies showed that CTGF-loaded Alg and AlgSulf2.0/PCL NPs result in the fastest and highest wound closure at the early and late stages of wound healing, respectively (36.49%, P<0.001 on day 1; 90.45%, P<0.05 on day 10), outperforming free CTGF. Double-emulsion NPs based on Alg or AlgSulf represent a viable strategy for delivering heparin-binding GF and other therapeutics, potentially aiding various disease treatments.

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

BackgroundTacrolimus (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 resultsThirty-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-β1) 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.ConclusionLina ameliorated TAC-induced kidney injury through modulation of oxidative stress, hypoxia, and apoptosis related proteins.

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 β1 (TGF-β1) 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.

التنبؤ المبكر بالاعتلال الكلوي لمرضى عراقين مصابين بالسكري النوع الثاني بدلالة تقييم بعض العوامل البيوكيمائية ذات الصلة

داء السكري من النوع 2 (T2DM) هو مرض معقد يصيب العديد من الأعضاء إلى جانب البنكرياس مثل الكلى والكبد والدماغ والعين. بسبب ارتفاع السكر في الدم لفترات طويلة وعدم السيطرة على مرض السكري مع وجود عوامل خطر أخرى ، يمكن أن تحدث مضاعفات مرض السكري. تشمل مضاعفات مرض السكري مضاعفات الأوعية الدموية الدقيقة والأوعية الدموية الكبيرة التي تستهدف الكلى. تهدف الدراسة الحالية إلى التحقق من مستوى الانجوتينسن المحول للانزيم ACE-2)) ، عامل نمو النسيج الضام (CTGF) جنبًا إلى جنب مع بعض العوامل البيوكيميائية ذات الصلة في النساء المصابات بداء السكري واعتلال الكلية السكري مقارنة بالضوابط الصحية. اشتملت الدراسة على 90 مريض تتراوح أعمارهم بين 30-65 سنة. يعانون من مرض السكري من النوع 2 مقسمون إلى ثلاث مجاميع على أساس معايير ACR تشمل البيلة الألبومينية الطبيعية ، البيلة الألبومينية الدقيقة ، البيلة الألبومينية الكبيرة 30 مريضًا لكل مجموعة و 30 شخصًا يتمتعون بصحة جيدة كانوا بمثابة المجموعة الضابطة ، منمن زاروا مستشفى بغداد التعليمي / المدينة الطبية و مستشفى اليرموك التعليمي ، في الفترة ما بين ديسمبر 2021 ومايو 2022. تم تحديد مستوياتACE-2 و CTGF باستخدام تقنية ELISA. أظهرت النتائج وجود فروق ذات دلالة إحصائية بين مجموعات المرضى والمجموعة الضابطة لعامل CTGF ، ووجد أن مستويات ACE-2 تزيد بشكل كبير في مجموعات المرضى عن مجموعة التحكم الصحية. كما أظهرت النتائج أن كلا من سكر الدم الصائم FBS و هيموكلوبينA1C (HbA1C)قد زاد بشكل ملحوظ في مجموعات المرضى مقارنة بالمجموعة الصحية. علاوة على ذلك ، كشفت قيم معدل الترشيح الكبيبي المقدر( ( eGFR عن فروق ذات دلالة إحصائية بين جميع المجموعات المدروسة ، وكذلك اظهرت نتائج ACR وجود فروقات ذات دلالة إحصائية بين مجموعات المرضى الثلاثة ، والتي تمثل المعايير الأساسية لتصنيف مجموعات المرضى. على أساس النتائج التي تم الحصول عليها في هذه الدراسة ، يمكن استنتاج ان عاملي ACE-2 و CTGF يمكن تطبيقهما كواسمات تنبؤية مبكرة موثوقة للكشف عن المرض.

Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Hypertrophic scarring (HS) is a pathological condition characterized by excessive fibrosis and inflammation, resulting in excessive extracellular matrix formation in the skin. MIR155HG, a long non-coding RNA, is abnormally upregulated in fibrotic tissues; however, its underlying mechanism is poorly understood. Using single-cell sequencing data, we analyzed connective tissue growth factor (CTGF) expression in various cell types in HS and normal skin tissues and MIR155HG expression in clinical samples. To investigate the mechanism of fibrosis, an in vitro model using CTGF-treated hypertrophic scar fibroblasts (HSFBs) was established and qRT-PCR, western blotting and ELISA assays were performed to investigate the expression of interleukin (IL)-1β, IL-6, and mesenchymal markers α-smooth muscle actin (α-SMA). CTGF stimulates MIR155HG level through phosphorylated STAT3 binding to the MIR155HG promoter. We analyzed the methylation of MIR155HG, assessed the levels of miR-155-5p/−3p in CTGF-treated HSFBs and identified differentially expressed genes among HS and NS samples using the Gene Expression Omnibus RNA sequencing data. The binding between miR-155-5p/−3p and AZGP1 was confirmed using a dual-luciferase assay and inflammatory cytokine production and α-SMA expression were investigated in rescue experiments. The findings revealed that CTGF elevated inflammatory cytokine production, α-SMA and MIR155HG expression in HSFBs. MIR155HG is upregulated in HS tissues due to low DNA methylation. Mechanistically, miR-155-5p/−3p was directly bound to MIR155HG 3’UTR. MIR155HG silencing inhibited cytokine production and α-SMA expression by repressing the generation of miR-155-5p/−3p in CTGF-treated HSFBs. Bioinformatics analysis and luciferase reporter assays revealed that miR-155-5p/−3p targets AZGP1. In addition, transfection with plasmids carrying AZGP1 cDNA significantly inhibited the signaling activity of miR-155-5p/−3 p-overexpressing HSFBs. Our findings highlight the importance of the MIR155HG/miR-155/AZGP1 axis in regulating cytokine production and α-SMA in HS.

The natural history of CVB3 myocarditis in C57BL/6J mice: an extended in-depth characterization

Background and aimsViral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course. Methods and resultsC57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 105 (regular dose)(RD) or 5 × 106 (high dose)(HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8 and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<0.001). In male CVB mice, premature mortality occurred between days 8-23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups. ConclusionCVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease.

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

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)-β. 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-β-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). 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-β-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. 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-β-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-β-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-β-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-β-treated NHLFs. TP213 alleviated TGF-β-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-β-induced p65 and α-SMA expression in NHLFs. 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.

The Role of Collagen VIII in the Aging Mouse Kidney.

The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-β1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.

Connective Tissue Growth Factor: Regulation, Diseases, and Drug Discovery.

In drug discovery, selecting targeted molecules is crucial as the target could directly affect drug efficacy and the treatment outcomes. As a member of the CCN family, CTGF (also known as CCN2) is an essential regulator in the progression of various diseases, including fibrosis, cancer, neurological disorders, and eye diseases. Understanding the regulatory mechanisms of CTGF in different diseases may contribute to the discovery of novel drug candidates. Summarizing the CTGF-targeting and -inhibitory drugs is also beneficial for the analysis of the efficacy, applications, and limitations of these drugs in different disease models. Therefore, we reviewed the CTGF structure, the regulatory mechanisms in various diseases, and drug development in order to provide more references for future drug discovery.

Design, Synthesis, and Bioevaluation of Transcriptional Enhanced Assocciated Domain (TEAD) PROTAC Degraders.

Dysregulation of the Hippo pathway has been observed in various cancers. The transcription factor TEAD, together with its coactivators YAP/TAZ, plays a crucial role in regulating the transcriptional output of the Hippo pathway. Recently, extensive research has focused on small molecule inhibitors targeting TEAD, but studies on TEAD degraders are comparatively rare. In this study, we designed and synthesized a series of TEAD PROTACs by connecting a pan-TEAD inhibitor with the CRBN ligand thalidomide. A representative compound, 27, exhibited potent antiproliferative activity against NF2-deficient NCI-H226 cells. It dose-dependently induced TEAD degradation dependent on CRBN and proteasome system and decreased key YAP target genes CYR61 and CTGF expressions in NCI-H226 cells. Further degradation selectivity studies revealed that 27 exhibited more potent activity against TEAD2 compared to those of the other three family members in Flag-TEADs transfected 293T cells. Therefore, 27 may serve as a valuable tool for advancing biological studies related to TEAD2.

CCN2/CTGF expression does not correlate with fibrosis in myeloproliferative neoplasms, consistent with noncanonical TGF-β signaling driving myelofibrosis.

The classical BCR::ABL1-negative myeloproliferative neoplasms (MPN) form a group of bone marrow (BM) diseases with the potential to progress to acute myeloid leukemia or develop marrow fibrosis and subsequent BM failure. The mechanism by which BM fibrosis develops and the factors that drive stromal activation and fibrosis are not well understood. Cellular Communication Network 2 (CCN2), also known as CTGF (Connective Tissue Growth Factor), is a profibrotic matricellular protein functioning as an important driver and biomarker of fibrosis in a wide range of diseases outside the marrow. CCN2 can promote fibrosis directly or by acting as a factor downstream of TGF-β, the latter already known to contribute to myelofibrosis in MPN.To study the possible involvement of CCN2 in BM fibrosis in MPN, we assessed CCN2 protein expression by immunohistochemistry in 75 BM biopsies (55 × MPN and 20 × normal controls). We found variable expression of CCN2 in megakaryocytes with significant overexpression in a subgroup of 7 (13%) MPN cases; 4 of them (3 × essential thrombocytemia and 1 × prefibrotic primary myelofibrosis) showed no fibrosis (MF-0), 2 (1 × post-polycythemic myelofibrosis and 1 × primary myelofibrosis) showed moderate fibrosis (MF-2), and 1 (primary myelofibrosis) severe fibrosis (MF-3). Remarkably, CCN2 expression did not correlate with fibrosis or other disease parameters such as platelet count or thrombovascular events, neither in this subgroup nor in the whole study group. This suggests that in BM of MPN patients other, CCN2-independent pathways (such as noncanonical TGF-β signaling) may be more important for the development of fibrosis.

Assembly of Interfacial Polyelectrolyte Complexation Fibers with Mineralization Gradient for Physiologically-Inspired Ligament Regeneration.

Current synthetic grafts for ligament rupture repair often fail to integrate well with the surrounding biological tissue, leading to complications such as graft wear, fatigue, and subsequent re-rupture. To address this medical challenge, this study aims at advancing the development of a biological ligament through the integration of physiologically-inspired principles and tissue engineering strategies. In this study, interfacial polyelectrolyte complexation (IPC) spinning technique, along with a custom-designed collection system, to fabricate a hierarchical scaffold mimicking native ligament structure, is utilized. To emulate the bone-ligament interface and alleviate stress concentration, a hydroxyapatite (HAp) mineral gradient is strategically introduced near both ends of the scaffold to enhance interface integration and diminish the risk of avulsion rupture. Biomimetic viscoelasticity is successfully displayed to provide similar mechanical support to native ligamentous tissue under physiological conditions. By introducing the connective tissue growth factor (CTGF) and conducting mesenchymal stem cells transplantation, the regenerative potential of the synthetic ligament is significantly amplified. This pioneering study offers a multifaceted solution combining biomimetic materials, regenerative therapies, and advanced techniques to potentially transform ligament rupture treatment.

Prognostic significance of connective tissue growth factor expression in stromal cells in patients with diffuse‑type gastric cancer.

Connective tissue growth factor (CTGF) is a target gene of the Hippo signaling pathway. Its differential role in the histological types of gastric cancer (GC) remains unknown; therefore, the present study aimed to confirm the clinical significance of CTGF expression in cancer and stromal cells in patients with GC depending on the histological type. The present study enrolled 589 patients with GC. Immunohistochemistry was used to analyze CTGF expression in cancer and stromal cells. CTGF mRNA expression data and the corresponding clinical information of GC samples were collected from The Cancer Genome Atlas (TCGA) database. Subsequently, the associations between CTGF expression and several clinicopathological factors were investigated. In the present study, CTGF expression was mainly observed in the cytoplasm of cancer and stromal cells. CTGF expression in stromal cells was significantly associated with CTGF expression in cancer cells (P<0.001). CTGF positivity in stromal cells was also significantly associated with intestinal type, non-scirrhous type, tumor depth (T1-2), lymph node metastasis (negative), lymphatic invasion (negative) and tumor size (<5 cm). Low CTGF expression in stromal cells was independently associated with worse overall survival (OS). Furthermore, the OS of patients with low CTGF expression in stromal cells, especially in patients with diffuse-type GC, was significantly worse than patients with high CTGF expression (P=0.022). This trend was similar to that revealed by TCGA data analysis. In conclusion, low CTGF expression was associated with a significantly worse OS in patients with diffuse-type GC. These data indicated that CTGF, and its control by the Hippo pathway, may be considered potential treatment targets in diffuse-type GC.

Diverse potential of secretome from natural killer cells and monocyte-derived macrophages in activating stellate cells.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-β1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor β1/Smad (TGF-β1/Smad) signaling pathway. Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway. A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-β signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-β2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-β2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-β2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-β2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Abstract 3016: MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development

Abstract Introduction: MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein), also known as Regnase-1, belongs to the CCCH zinc finger protein family, featuring a PilT N-terminus (PIN) domain-like RNase domain. Utilizing its intrinsic endonuclease activity, MCPIP1 functions as a precise modulator of cytokines, exhibiting an anti-inflammatory effect through the targeted degradation of specific mRNA transcripts. While recent studies have highlighted MCPIP1 as a potential tumor suppressor with decreased levels in breast cancer and clear cell renal cell carcinoma, its role in hepatocellular carcinoma (HCC) development remains unexplored. Material and Methods: To investigate the role of MCPIP1 in HCC development, we used tissue-specific knockout mice with the deletion of Mcpip1 in hepatocytes. HCC was induced by administering diethylnitrosamine (DEN) to two-week-old mice. We conducted a comprehensive analysis, including the assessment of EMT and fibrosis markers, proinflammatory cytokines, and immune cell infiltration, using Western blot (WB), mass spectrometry, quantitative PCR (qPCR), and multiple immunohistochemistry (IHC) stainings. To identify differences between the wild-type (wt) and Mcpip1 knockout (KO) mice groups more effectively, hepatocytes were isolated using the perfusion method, and RNA-Seq analysis was performed. Results and Discussion: The tissue-specific knockout of Mcpip1 in the liver has notable molecular consequences. Transcriptomic profiling revealed an upregulation of transcripts integral to tumor development, including Cxcr4, Met, Cxcl12, Hgf, and Src, alongside factors characteristically associated with cancer stem cells, Myc, Klf4, and CD133. The absence of MCPIP1 triggered immune cell activation in the liver, as evidenced by an augmented expression of markers specific to myeloid and lymphoid cell populations, indicative of an intensified immunological response. IHC examination of liver tissue from Mcpip1-deficient mice revealed significant fibrotic alterations, characterized by increased collagen deposition, and enhanced expression of alpha-smooth muscle actin (αSMA). The lack of Mcpip1 influences the increased levels of vimentin, fibronectin, and connective tissue growth factor (cTGF), indicating enhanced tissue remodeling. Conclusions: MCPIP1, recognized for its anti-inflammatory functions, appears to function as a potential tumor suppressor in HCC development. The lack of MCPIP1 results in the upregulation of transcripts linked to tumorigenesis, immune cell activation, and notable fibrotic changes, highlighting its involvement in the complex molecular dynamics of HCC progression. This study was supported by National Science Center grants no. 2017/26/E/NZ5/00691, 2021/41/N/NZ4/04187. Citation Format: Katarzyna Miekus, Oliwia Kwapisz, Paulina Marona, Judyta Gorka, Jerzy Kotlinowski, Ewelina Pospiech, Jolanta Jura. MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3016.

Exosomal-miRNas expression and growth factors released by mononuclear cells of CLAD patients in response to extracorporeal photopheresis

BackgroundCLAD (Chronic Lung Allograft Dysfunction) remains a serious complication following lung transplantation. Some evidence shows that portions of Extracorporeal Photopheresis (ECP)-treated patients improve/stabilize their graft function. In spite of that, data concerning molecular mechanisms are still lacking. Aims of our study were to assess whether ECP effects are mediated by Mononuclear Cells (MNCs) modulation in term of microRNAs (miRNAs) expression and growth factors release.MethodsCells from leukapheresis of 16 CLAD patients, at time 0 and 6-months (10 cycles), were cultured for 48h ± PHA (10 ug/ml) or LPS (2 ug/ml). Expression levels of miR-146a-5p, miR-155-5p, miR-31-5p, miR181a-5p, miR-142-3p, miR-16-5p and miR-23b-5p in MNCs-exosomes were evaluated by qRT-PCR, while ELISA assessed different growth factors levels on culture supernatants.ResultsOur result showed miR-142-3p down-regulation (p = 0.02) in MNCs of ECP-patients after the 10 cycles and after LPS stimulation (p = 0.005). We also find miR-146a-5p up-regulation in cells after the 10 cycles stimulated with LPS (p = 0.03). Connective tissue growth factor (CTGF) levels significantly decreased in MNCs supernatant (p = 0.04). The effect of ECP is translated into frequency changes of Dendritic Cell (DC) subpopulations and a slight increase in T regulatory cells (Treg) number and a significant decrease in CTGF release.ConclusionsECP might affect regulatory T cell functions, since both miR-142 and miR-146a have been shown to be involved in the regulation of suppressor regulatory T cell functions and DCs. On the other side ECP, possibly by regulating macrophage activation, is able to significantly down modulate CTGF release.