Connective Tissue Growth Factor Levels Research Articles

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.

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).

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.

Galectin-3 promotes fibrosis in ovarian endometriosis.

This study aimed to investigate the potential role of galectin-3 (Gal-3) in the pathogenesis of fibrotic alterations in ovarian endometriosis (OVE). In this study, we collected the ectopic endometrial tissues and eutopic endometrial tissues from 31 OVE patients treated by laparoscopy, and the eutopic endometrial tissues from 23 non-OVE patients with leiomyoma or other benign diseases were used as control. Hematoxylin and eosin (H&E) and Masson's trichrome staining were utilized for histopathological assessment. The primary normal endometrial stromal cells (NESC), ectopic endometrial stromal cells (ECSC), and eutopic endometrial stromal cells (EUSC) were isolated. Gal-3 overexpression plasmids (Gal-OE) and short hairpin RNA targeting Gal-3 (Gal-3-shRNA) were transfected into the immortalized human endometriotic cell line 12Z, respectively. RT-qPCR, Western blot analysis, and immunohistochemistry were used to detect the mRNA and protein expression levels of Gal-3, type I collagen (COL-1), connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA), respectively. H&E and Masson staining showed that ovarian ectopic endometrium exhibited glandular hyperplasia, high columnar glandular epithelium, apical plasma secretion, more subnuclear vacuoles, and obvious fibrosis, compared with normal endometrium. The mRNA and protein levels of Gal-3 , CTGF, α-SMA, and COL-1 were all upregulated in the ectopic endometrial tissues of OVE patients compared to the eutopic endometrial tissues from OVE patients and non-OVE patients. Moreover, ECSC expressed higher levels of Gal-3, CTGF, α-SMA, and COL-1 than EUSC and NESC. Follow-up investigations demonstrated that the Gal-3 overexpression substantially increased fibrosis-related markers including CTGF, α-SMA, and COL-1 within the 12Z cell line. Conversely, Gal-3 knockdown showed the opposite effects. Gal-3 promotes fibrosis in OVE, positioning it as a prospective therapeutic target for mitigating fibrosis in endometriosis.

The association of plasma connective tissue growth factor levels with left ventricular diastolic dysfunction in patients with overt hyperthyroidism.

Left ventricular (LV) diastolic dysfunction is an independent predictor of future cardiovascular events. Early detection of patients with LV diastolic dysfunction can improve clinical outcomes through active management. However, the assessment of diastolic function is very complicated, and there are currently lack of effective biomarkers to assess the risk of LV diastolic dysfunction. Connective tissue growth factor (CTGF) plays a significant role in cardiac remodeling and dysfunction. We aimed to investigate the associations between plasma CTGF level and the risk of LV diastolic dysfunction in this study and judge its effectiveness in diagnosing LV diastolic dysfunction. A total of 169 patients with overt hyperthyroidism were included. LV diastolic function was evaluated and the subjects were divided into normal LV diastolic function group and LV diastolic dysfunction group. Routine clinical medical data, biochemical data, thyroid related parameters and echocardiographic parameters were recorded for analysis. Compared with normal LV diastolic function group, the LV diastolic dysfunction group had higher age and BMI, as well as lower heart rate, lower serum albumin, lower eGFR, higher serum TgAb and BNP level, and the incidences of hypertension were also higher (all P <0.05). Circulating plasma CTGF levels in the LV diastolic dysfunction group were significantly higher (normal LV diastolic function group: 7.026 [5.567-8.895], LV diastolic dysfunction group: 8.290 [7.054-9.225] ng/ml, median [(Interquartile range)], P = 0.004); Compared with the lowest quartile group, the crude odds ratios (OR) of LV diastolic dysfunction in the second, third, and fourth quartile group were 3.207, 5.032 and 4.554, respectively (all P<0.05). After adjustment for the potentially confounding variables, the adjusted OR values of the third and fourth quartile group had no obvious change. The results of ROC showed that the plasma CTGF had the largest area under the ROC curve, and the value was 0.659 (P = 0.005). The level of circulating plasma CTGF in the LV diastolic dysfunction group was significantly increased. Plasma CTGF level is an independent risk factor for LV diastolic dysfunction. Compared with serum BNP level, the plasma CTGF level may have auxiliary diagnostic value for LV diastolic dysfunction in hyperthyroid patients.

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.

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.

Cardiac-specific deletion of myosin phosphatase targeting subunit 2 alleviates cardiac fibrosis in mineralocorticoid receptor-related hypertension

Abstract Introduction Myosin phosphatase targeting subunit 2 (MYPT2) is a crucial subunit of cardiac myosin light chain (MLC) phosphatase. The phosphorylation of MLC in the ventricle plays a pivotal role in cardiac hypertrophy and function. Our previous study demonstrated that overexpression of MYPT2 activated MLC phosphatase leading to cardiac dysfunction. Mineralocorticoid receptor (MR)-related hypertension is associated with extensive cardiac fibrosis. It remains unknown whether MYPT2 may play a role in the development of cardiac fibrosis in MR-related hypertension. Purpose Our purpose is to investigate the effect of MYPT2 on cardiac fibrosis in MR-related hypertension. Methods After the knockdown of MYPT2 using MYPT2-siRNA, HL-1 cells (murine cardiomyocytes) were incubated with aldosterone (ALDO) for 24h. Cardiac-­specific MYPT2 knockout (c-MYPT2-/-) mice were generated using the Cre-lox system (αMHC-­Cre; MYPT2f/f). MR-related hypertension mice were induced in c-MYPT2-/- and wild-type (MYPT2+/+) mice with the subcutanouse infusion of ALDO and 8% NaCl food for 4 weeks after uninephrectomy. Systolic blood pressure (SBP) was measured by tail cuffs every week. WB, qPCR, cardiac echo, and histological examinations were performed. Results In HL-1 cells, ALDO increased the expression of connective tissue growth factor (CTGF), MYPT2, and the catalytic subunit of the type-1 phosphatase δ isoform (PP1cδ) in a concentration-dependent manner. MYPT2 protein levels significantly decreased by 80% in HL-1 cells transfected with MYPT2 siRNA compared with cells transfected with unspecific siRNA. Knockdown of MYPT2 significantly decreased the protein level of CTGF. In the mice model, the MYPT2 expression in the heart from MYPT2-/- mice was decreased to less than 30% compared with MYPT2+/+ mice. Heart rate, blood pressure, and cardiac systolic function were normal in c-MYPT2-/- mice. PP1cδ was decreased in c-MYPT2-/- mice compared with MYPT2+/+ mice. The expression of cardiac MLC kinase was not changed. The level of phosphorylation of MLC in c-MYPT2-/- mice was significantly higher than MYPT2+/+. Blood pressure elevation and left ventricular hypertrophy were observed in both MR-related hypertension mice. No significant difference in heart size and the nuclear localization of MR in cardiomyocytes. However, ejection fraction and fractional shortening in echocardiography were higher in c-MYPT2-/- than in MYPT2+/+ in MR-related hypertension mice. Histopathological examinations revealed the degree of fibrosis in c-MYPT2-/- mice was lower than in MYPT2+/+ mice with the decreased expression of CTGF. Conclusions Cardiac-specific deletion of MYPT2 resulted in a decrease of MLC phosphatase and an increase of phosphorylation in MLC in vivo. MYPT2 deletion can inhibit cardiac fibrosis in a MR-related hypertension model.

Pharmacokinetic-pharmacodynamic modeling of the active components of Shenkang injection in rats with chronic renal failure and its protective effect on damaged renal cells.

The study aimed to explore the pharmacokinetic and pharmacodynamic alterations of the active components of Shenkang injection (i.e. hydroxy saffron yellow pigment A [HSYA], tanshinol, rheum emodin, and astragaloside IV) in rats with chronic renal failure (CRF), and establish a pharmacokinetic-pharmacodynamic model (PK-PD model) in order to provide a scientific and theoretical basis for the rational clinical use of Shenkang injection. Sprague-Dawley (SD) rats were randomly divided into a normal group, model group, and Shenkang injection group. A rat model of CRF was induced by adenine gavage and then followed by drug administration via tail vein injection. Orbital blood was collected at different timepoints and the blood concentrations of the four active components were measured by UHPLC-Q-Orbitrap HRMS. Serum levels of creatinine (Scr), urea nitrogen (BUN), and uric acid (UA) were determined using an automatic biochemical analyzer. A PK-PD model was established, and DAS 3.2.6 software was used for model fitting as well as statistical analysis. TGF-β1 was utilized to induce normal rat kidney cells to construct a renal fibrosis model to investigate the protective effect of the pharmacological components on renal fibrosis. The pharmacokinetic analysis of hydroxy saffron yellow pigment A, tanshinol, rheum emodin, and astragaloside IV based on UHPLC-Q-Orbitrap HRMS was stable. The linear regression equations for the four active components were as follows: Y=0.031X+0.0091 (R2 =0.9986) for hydroxy saffron yellow pigment A, Y=0.0389X+0.164 (R2 =0.9979) for tanshinol, Y=0.0257X+0.0146 (R2 =0.9973) for rheum emodin, and Y=0.0763X+0.0139 (R2 =0.9993) for astragaloside IV, which indicated good linear relationships. The methodological investigation was stable, with the interday and intraday precision RSD <10%. Meanwhile, the recoveries ranged between 90% and 120%, in accordance with the requirements for in vivo analysis of drugs. Compared with the model group, the levels of Scr, BUN, and UA were significantly decreased after 20min in the Shenkang injection group (p<0.01). The PK-PD model showed that the four active components in the Shenkang injection group could fit well with the three effect measures (i.e. Scr, BUN, and UA), with the measured values similar to the predicted values. The cell model of renal fibrosis showed that the connective tissue growth factor and FN1 protein expression levels were significantly lower in the Shenkang injection group than those in the model group, and the cell fibrosis was improved. The established method for in vivo analysis of Shenkang injection was highly specific, with good separation of the components and simple operation. The total statistical moment could well integrate the pharmacokinetic parameters of the four active components. After treatment with Shenkang injection, all indexes in the administered group improved and showed significant inhibition of renal cell fibrosis in vitro. This study could provide scientific reference ideas for the clinical rational use of traditional Chinese medicine.

Irpex lacteus polysaccharide exhibits therapeutic potential for ovarian fibrosis in PCOS rats via the TGF-β1/smad pathway

Polycystic ovarian syndrome (PCOS) is one of the commonest endocrinopathies in childbearing women. The research was conducted to assess the impact of Irpex lacteus polysaccharide (ILP, 1000 mg/kg) on the letrozole (1 mg/kg)-induced PCOS model in female rats. Metformin (Met, 265 mg/kg) as the positive control. The study suggested that ILP restored the estrous cycle in rats with PCOS as well as lowered relative ovarian weight and body weight, in comparison to normal. Rats with PCOS showed improvement in ovarian structure and fibrosis when given ILP. ILP decreased the testosterone (T), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), total cholesterol (TC), luteinizing hormone (LH), homeostasis model assessment-insulin resistance (HOMA-IR), fasting blood glucose (FBG), and insulin (INS) levels and elevated the follicle-stimulating hormone (FSH) and estrogen (E2) levels in PCOS rats. In addition, ILP increased the content of superoxide dismutase (SOD) in serum and the antioxidant enzymes (Prdx3, Sod1, Gsr, Gsta4, Mgst1, Gpx3, Sod2 and Cat) expression levels in the ovaries and decreased the serum expression of malondialdehyde (MDA). In addition, ILP treatment slowed down the process of the fibrosis-associated TGF-β1/Smad pathway and downregulated α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) levels in PCOS rats ovaries. According to these findings, ILP may be able to treat letrozole-induced PCOS in rats by ameliorating metabolic disturbances, sex hormone levels, oxidative stress, and ovarian fibrosis.

Sodium hyaluronate promotes proliferation, autophagy, and migration of corneal epithelial cells by downregulating miR-18a in the course of corneal epithelial injury

Corneal epithelium can resist the invasion of external pathogenic factors to protect the eye from external pathogens. Sodium hyaluronate (SH) has been confirmed to promote corneal epithelial wound healing. However, the mechanism by which SH protects against corneal epithelial injury (CEI) is not fully understood. CEI model mice were made by scratching the mouse corneal epithelium, and in vitro model of CEI were constructed via curettage of corneal epithelium or ultraviolet radiation. The pathologic structure and level of connective tissue growth factor (CTGF) expression were confirmed by Hematoxylin and Eosin staining and immunohistochemistry. CTGF expression was detected by an IHC assay. The levels of CTGF, TGF-β, COLA1A, FN, LC3B, Beclin1, and P62 expression were monitored by RT-qPCR, ELISA, Western blotting or immunofluorescence staining. Cell proliferation was detected by the CCK-8 assay and EdU staining. Our results showed that SH could markedly upregulate CTGF expression and downregulate miR-18a expression in the CEI model mice. Additionally, SH could attenuate corneal epithelial tissue injury, and enhance the cell proliferation and autophagy pathways in the CEI model mice. Meanwhile, overexpression of miR-18a reversed the effect of SHs on cell proliferation and autophagy in CEI model mice. Moreover, our data showed that SH could induce the proliferation, autophagy, and migration of CEI model cells by downregulating miR-18a. Down-regulation of miR-18a plays a significant role in the ability of SH to promote corneal epithelial wound healing. Our results provide a theoretical basis for targeting miR-18a to promote corneal wound healing.

The effect of ET1-CTGF mediated pathway on the accumulation of extracellular matrix in the trabecular meshwork and its contribution to the increase in IOP.

To investigate the effect of endothelin-1 (ET-1) in excessive accumulation of extracellular matrix (ECM) of the trabecular meshwork (TM) and its role in intraocular pressure (IOP) regulation. Cultured human TM cells (HTMCs) were treated with ET-1, ET-1 + ETA receptor (ETAR) antagonist BQ123, ET-1 + ETB receptor (ETBR) antagonist BQ788. The expressions of fibronectin (FN) and collagen type IV (Col IV) were evaluated by western blotting and immunofluorescence. A time course effect of ET-1 on the transcription level of connective tissue growth factor (CTGF) was investigated by qRT-PCR. Next, the transcription level of CTGF was downregulated by using antisense oligodeoxynucleotide sequence. Then HTMCs were treated with ET-1, and the expression levels of FN and Col IV were evaluated by western blotting. In addition, by using an ex-vivo model of cultured anterior eye segment, we explored the effect of ET-1 on IOP changes and the expressions of FN and Col IV. In cultured HTMCs, the expressions of FN and Col IV were significantly increased after ET-1 treatment, which were blocked by the pretreatment of ETAR antagonist BQ123, rather than ETBR antagonist BQ788. Besides, the CTGF mRNA level increased significantly and reached a peak after 48h of ET-1 treatment. However, the effect of ET-1 on increasing the expressions of FN and Col IV in HTMCs could be inhibited by the downregulation of CTGF. In an ex-vivo model, IOP increased significantly after ET-1 administration, which could be blocked by BQ123 but not by BQ788. Furthermore, elevated expressions of FN and Col IV in TM were observed after ET-1 perfusion, and could be inhibited by BQ123 pretreatment. Excessive ET-1 in aqueous humor could lead to the abnormal accumulation of FN and Col IV in TM via the ETA-CTGF pathway, thereby increasing IOP.

Role of mechanotransduction mediated by YAP/TAZ in the treatment of neurogenic erectile dysfunction with low-intensity pulsed ultrasound.

Erectile dysfunction (ED) and weakness of the penis are processes related to hemodynamic alteration. Low-intensity pulsed ultrasound (LIPUS), as a new mechanical modality for the treatment of ED, deserves to be explored in depth for the biomechanical mechanisms it exerts. The aim of this study was to explore the role of YAP/TAZ-mediated mechanotransduction in mechanical therapy for the treatment of neurogenic erectile dysfunction (NED). Forty-two male SD rats (12 w old) were randomly divided into sham-operated (n=14), bilateral cavernous nerve injury (BCNI, n=14), and LIPUS-treated (n=14) groups. Intracavernosal pressure/mean arterial pressure (ICP/MAP) was measured 14 and 28 days after treatment. Penile tissue specimens were collected for pathological examination, and the changes in YAP, TAZ, connective tissue growth factor (CTGF), CYR61, LATS1, and p38 mitogen-activated protein kinase expression levels were assessed by Western blot, real-time quantitative polymerase chain reaction (RT-qPCR) and immunological staining. Compared with BCNI, LIPUS significantly improved ICP/MAP levels and enhanced histopathological changes. The penile expression levels of YAP, TAZ, CTGF, and CYR61 were significantly downregulated in the BCNI group (p<0.01), and LIPUS upregulated the expression levels of these proteins (p<0.05). The expression levels of p-LATS1 and LATS1 were not significantly different among the groups (p>0.05). Interestingly, the expression level of p-p38/p38 significantly increased in BCNI rats (p<0.05), which was reversed by LIPUS treatment (p<0.05). However, the p38 inhibitor SB203580 did not change the expression of YAP/TAZ in rat primary smooth muscle cells or mouse MOVAS cells (p>0.05). LIPUS can effectively improve penile erectile function in NED rats. The underlying mechanism may be related to the regulation of YAP/TAZ-mediated mechanotransduction. However, the upstream regulatory signal may differ from the classical Hippo pathway.

Abstract 431: FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor

Abstract Metastatic melanoma is one of the most aggressive skin cancers and is associated with poor prognosis. BRAF and MEK inhibitors are used to treat patients with BRAFV600E-mutated advanced melanoma. However, the development of resistances to these treatments compromises therapeutic success. Our lab previously demonstrated that forkhead box D1 (FOXD1) plays a critical role in melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells. Immunohistochemical assessment of 105 samples from patients with metastatic melanoma revealed that high FOXD1 expression in tumors was associated with poor survival and correlated with low MITF, SOX10 and high AXL expression. Upregulation of FOXD1 expression enhanced the resistance of melanoma to vemurafenib (BRAF inhibitor) or combinatorial treatment with vemurafenib and cobimetinib (MEK inhibitor). On the other hand, loss of FOXD1 increased the sensitivity of naïve melanoma cells towards vemurafenib or combinatorial treatment with vemurafenib and cobimetinib. Furthermore, high FOXD1 expression levels were found in BRAF inhibitor (BRAFi)-resistant cells. Downregulation of FOXD1 resulted in a resensitization of BRAFi-resistant cells to vemurafenib. By using microarray analysis, connective tissue growth factor (CTGF) was found to be one of the most downregulated genes in FOXD1 knockdown (KD) cells while its expression was highly increased upon FOXD1 overexpression. Thus, CTGF was identified as a downstream factor of FOXD1. In addition, in vitro expression analysis and evaluation of clinical samples demonstrated that CTGF and FOXD1 expression were positively correlated. By using a CHIP assay and a dual reporter luciferase assay, we discovered that FOXD1 could regulate the expression of CTGF by directly binding to the CTGF promoter. This result was confirmed with RT-PCR and western blot. In addition, we found that the protein level of CTGF was highly increased in BRAFi-resistant cells. Similar to FOXD1 knockdown, the knockdown of CTGF resensitized BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that the transcription factor FOXD1 could promote dedifferentiation and targeted therapy-resistance in melanoma cells by regulating the expression of CTGF. FOXD1 might be a promising new diagnostical marker and a therapeutic target of targeted therapy resistant melanoma. Citation Format: Qian Sun, Daniel Novak, Laura Hüser, Juliane Poelchen, Huizi Wu, Karol Granados, Aniello Federico Federico, Ke Liu, Tamara Steinfass, Marlene Vierthaler, Viktor Umansky, Jochen Utikal. FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 431.

The Role of CTGF in Liver Fibrosis Induced in 3D Human Liver Spheroids.

Connective tissue growth factor (CTGF) is involved in the regulation of extracellular matrix (ECM) production. Elevated levels of CTGF can be found in plasma from patients with liver fibrosis and in experimental animal models of liver fibrosis, but the exact role of CTGF in, e.g., diet-induced human liver fibrosis is not entirely known. To address this question, we utilized a 3D human liver co-culture spheroid model composed of hepatocytes and non-parenchymal cells, in which fibrosis is induced by TGF-β1, CTGF or free fatty acids (FFA). Treatment of the spheroids with TGF-β1 or FFA increased COL1A1 deposition as well as the expression of TGF-β1 and CTGF. Recombinant CTGF, as well as angiotensin II, caused increased expression and/or production of CTGF, TGF-β1, COL1A1, LOX, and IL-6. In addition, silencing of CTGF reduced both TGF-β1- and FFA-induced COL1A1 deposition. Furthermore, we found that IL-6 induced CTGF, COL1A1 and TGF-β1 production, suggesting that IL-6 is a mediator in the pathway of CTGF-induced fibrosis. Taken together, our data indicate a specific role for CTGF and CTGF downstream signaling pathways for the development of liver inflammation and fibrosis in the human 3D liver spheroid model.

Prolactin promotes crop epithelial proliferation of domestic pigeons (Columba livia) through the Hippo signaling pathway.

The purpose of this study was to investigate whether prolactin (PRL) regulates the proliferation of pigeon crop epithelium through the Hippo signaling pathway during the breeding cycle. Twenty-four pairs of adult pigeons were allotted to four groups by different breeding stages, and their crops and serum were sampled. Eighteen pairs of young pigeons were selected and divided into three groups for the injection experiments. The results showed that the serum PRL content and crop epithelial thickness of pigeons increased significantly at day 17 of incubation (I17) and day 1 of chick-rearing (R1). In males, the mRNA levels of yes-associated transcriptional regulator (YAP) and snail family transcriptional repressor 2 (SNAI2) were peaked at I17, and the gene levels of large tumor suppressor kinase 1 (LATS1), serine/threonine kinase 3 (STK3), TEA domain transcription factor 3 (TEAD3), connective tissue growth factor (CTGF), MYC proto-oncogene (c-Myc) and SRY-box transcription factor 2 (SOX2) reached the maximum value at R1. In females, the gene expression of YAP, STK3, TEAD3, and SOX2 reached the greatest level at I17, the expression profile of SAV1, CTGF, and c-Myc were maximized at R1. In males, the protein levels of LATS1 and YAP were maximized at R1 and the CTGF expression was upregulated at I17. In females, LATS1, YAP, and CTGF reached a maximum value at I17, and the expression level of phosphorylated YAP was minimized at I17 in males and females. Subcutaneous injection of prolactin (injected for 6 d, 10 μg per kg body weight every day) on the left crop of pigeons can promote the proliferation of crop epithelium by increasing the CTGF level and reducing the phosphorylation level of YAP. YAP-TEAD inhibitor verteporfin (injection for 6 d, 2.5 mg per kg body weight every day) can inhibit the proliferation of crop epithelium induced by prolactin by inhibiting YAP and CTGF expression. In conclusion, PRL can participate in crop cell proliferation of pigeons by promoting the expression of YAP and CTGF in Hippo pathway.

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

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

Comparison and Evaluation of CTGF and P2/MS as Noninvasive Markers in Fibrosis Evaluation in Chronic Liver Disease

Background/Aims: To compare CTGF (connective tissue growth factor) and P2/MS [platelet count (109 / L)] 2 / [monocyte fraction (%) x segment neutrophil fraction (%)] ratio as non-invasive tests with liver biopsy in the detection of liver fibrosis. Thus, to determine whether CTGF and P2/MS index results can be used as a noninvasive marker, in the diagnosis of chronic liver disease &#x0D; Materials and Methods: A total of 52 chronic hepatitis patients with concurrent liver biopsy (27 female, 25 male) were included in the study group. The subjects' ages ranged between 18 and 70, and none of the subjects had undergone liver disease-related treatment. The control group was made up of a total of 31 healthy individuals without liver disease (18 female, 13 male) from similar age groups. Liver biopsy results of patients were reported at 7 stages according to Modified Knodell Scoring (ISHAK).&#x0D; Results: CTGF levels were higher in patients with chronic hepatitis (1.06±0.70 ng/ml) than the control group (0,72±0,32 ng/ml) (P

LncRNA NEAT1 accelerates renal fibrosis progression via targeting miR-31 and modulating RhoA/ROCK signal pathway.

Renal fibrosis is the final pathway for chronic kidney disease to end-stage renal failure. Noncoding RNAs have been reported to play a crucial role in renal fibrosis. Here, the effects of long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) and miR-31 on renal fibrosis and their regulatory mechanism were evaluated. RT-qPCR was used to assess NEAT1, miR-31, and RhoA levels. Western blot was performed to analyze the expression of fibrosis markers, RhoA, rho-related kinase (ROCK1), and connective tissue growth factor (CTGF). RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH), and luciferase reporter assays verified the interaction between miR-31 and NEAT1 or RhoA. Renal fibrosis and injury were observed by Masson and hematoxylin and eosin (H&E) staining. The expression level of inflammatory cytokines was detected by ELISA. Immunohistochemistry (IHC) was performed to examine the expression levels of α-smooth muscle actin (α-SMA) and RhoA in renal tissues. We showed that NEAT1 was highly expressed, whereas miR-31 was decreased in renal fibrosis. NEAT1 was found to directly bind miR-31 to positively regulate RhoA expression. Furthermore, NEAT1 silencing inhibited renal fibrosis and inflammation and suppressed the RhoA/ROCK1 signaling pathway. However, knockdown of miR-31 could reverse these effects. NEAT1 silencing or overexpression of miR-31 alleviated renal fibrosis in vivo. In conclusion, NEAT1 accelerates renal fibrosis progression via negative regulation of miR-31 and the activation of RhoA/ROCK1 pathway, thereby upregulating the expression level of CTGF, providing a theoretical basis for treatment and prognostic evaluation of renal fibrosis.

Endothelial cell‑derived connective tissue growth factor stimulates fibroblast differentiation into myofibroblasts through integrin αVβ3.

Connective tissue growth factor (CTGF) is expressed at high levels in blood vessels, where it functions as a regulator of a number of physiological processes, such as cell proliferation, angiogenesis and wound healing. In addition, CTGF has been reported to be involved in various pathological processes, such as tumor development and tissue fibrosis. However, one of the main roles of CTGF is to promote the differentiation of fibroblasts into myofibroblasts, a process that is involved in disease progression. Therefore, the present study aimed to investigate the possible mechanism by which pathological changes in the microvasculature can direct the activation of fibroblasts into myofibroblasts in the context of hypoxia/reoxygenation (H/R). Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts were used in the present study. The expression levels of CTGF were determined by western blot analysis and reverse transcription-semi-quantitative PCR. To analyze the paracrine effect of HUVECs on fibroblasts, HUVECs were infected with CTGF-expressing adenovirus and then the culture supernatant of HUVECs was collected to treat fibroblasts. The formation of α-smooth muscle actin (α-SMA) stress fibers in fibroblasts were observed by immunofluorescence staining. It was found that H/R significantly increased CTGF expression in HUVECs. CTGF was also able to directly induce the differentiation of fibroblasts into myofibroblasts. In addition, the culture supernatant from CTGF-overexpressing HUVECs stimulated the formation of α-SMA stress fibers in fibroblasts, which was inhibited by treatment with a functional blocking antibody against integrin αVβ3 and to a lesser degree by a blocking antibody against α6 integrin. The mechanism of CTGF upregulation by H/R in HUVECs was then evaluated, where it was found that the CTGF protein was more stable in the H/R group compared with that in the normoxic control group. These findings suggest that CTGF expressed and secreted by vascular endothelial cells under ischemia/reperfusion conditions can exert a paracrine influence on neighboring fibroblasts, which may in turn promote myofibroblast-associated diseases. This association may hold potential as a therapeutic target.