Fibronectin Levels Research Articles

New triterpenoids from the Cyclocarya paliurus (Batalin) Iljinskaja and their anti-fibrotic activity

Cyclocarya paliurus, a Chinese herbal medicine and new food resource, contains a triterpenic-acid-rich extract that demonstrated ameliorative effect on diabetic nephropathy (DN). A more in-depth discovery of functional components led to the isolation of seven new triterpenoids including two pentacyclic triterpenes, 1α,2α,3β,23-tetrahydroxyolean-12-en-28-oic acid and 2α,3β,22α-tirhydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranoside, and five tetracyclic triterpenoid glycosides (cypaliurusides N-R), together with twelve known compounds from the leaves of C. paliurus. Their structures were determined using a comprehensive analysis of chemical and spectroscopic data. Partial compounds were assessed for anti-fibrotic activities in high-glucose and TGF-β1 induced HK-2 cells. Compound 16 remarkably decreased the level of fibronectin with an inhibition rate of 37.1%. Furthermore, 16 effectively alleviated the epithelial-mesenchymal transformation (EMT) process by upregulating E-cadherin expression and downregulating α-SMA expression, and it significantly decreased the level of the transcriptional inhibitors (Snail and Twist) of E-cadherin. The discovery of anti-fibrotic compounds from C. paliurus provides the potential utilization and functional candidates for the DN prevention.

Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of target genes. Therefore, the aim of this study was to determine the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-β. Five candidates HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the expression levels of fibronectin, vimentin and α-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined Ppia and Gapdh as the best HKG pair, and validation of the HKG by normalizing fibronectin, vimentin and α-SMA were consistent with results from the literature. Our results established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-β.

Fetal Fibronectin as a Predictor of Preterm Delivery: A Nigerian Cohort Study.

Background Fetal fibronectin is a useful biomarker in the diagnosis and management of preterm labour. Objectives To evaluate the relationship between cervical fetal fibronectin and preterm delivery and the association between cervical fetal fibronectin level and gestational age at delivery. Materials and Methods A prospective cohort study was performed in a tertiary hospital in Nigeria, involving equal number of pregnant women with (96) and without (96) preterm labour. Fetal fibronectin assay was done using solid-phase immunogold assay. The data were analysed using IBM SPSS version 24. Descriptive and inferential statistical analyses were done. The level of significance was p-value <0.05. Results Less than half (47.9%) of the women in the study group had preterm delivery while 13.09% of the women in the control group delivered preterm. Fetal fibronectin test had a sensitivity, specificity, positive predictive value and negative predictive value of 78%, 86.5%, 71.9%, and 89.0%, respectively, a positive likelihood ratio and negative likelihood ratio of 5.76(95% CI, 3.67 – 9.64) and 0.26(95% CI, 0.16 – 0.41), respectively. Conclusion The findings in our study value of fetal fibronectin in predicting preterm delivery. Its use will support less intervention for patients with negative results.

Lysophosphatidic Acid Promotes Epithelial-Mesenchymal Transition in Kidney Epithelial Cells via the LPAR1/MAPK-AKT/KLF5 Signaling Pathway in Diabetic Nephropathy.

The epithelial–mesenchymal transition (EMT) is a differentiation process associated with fibrogenesis in diabetic nephropathy (DN). Lysophosphatidic acid (LPA) is a small, naturally occurring glycerophospholipid implicated in the pathogenesis of DN. In this study, we investigated the role of LPA/LPAR1 signaling in the EMT of tubular cells as well as the underlying mechanisms. We observed a decrease in E-cadherin and an increase in vimentin expression levels in the kidney tubules of diabetic db/db mice, and treatment with ki16425 (LPAR1/3 inhibitor) inhibited the expression of these EMT markers. Ki16425 treatment also decreased the expression levels of the fibrotic factors fibronectin and alpha-smooth muscle actin (α-SMA) in db/db mice. Similarly, we found that LPA decreased E-cadherin expression and increased vimentin expression in HK-2 cells, which was reversed by treatment with ki16425 or AM095 (LPAR1 inhibitor). In addition, the expression levels of fibronectin and α-SMA were increased by LPA, and this effect was reversed by treatment with ki16425 and AM095 or by LPAR1 knockdown. Moreover, LPA induced the expression of the transcription factor, Krüppel-like factor 5 (KLF5), which was decreased by AM095 treatment or LPAR1 knockdown. The expression levels of EMT markers and fibrotic factors induced by LPA were decreased upon KLF5 knockdown in HK-2 cells. Inhibition of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and serine-threonine kinase (AKT) pathways decreased LPA-induced expression of KLF5 and EMT markers. In conclusion, these data suggest that LPA contributes to the pathogenesis of diabetic nephropathy by inducing EMT and renal tubular fibrosis via regulation of KLF5 through the LPAR1.

Effects of cyanate on pulmonary epithelial cells and pulmonary function in mice

To investigate the injury of cyanate on the pulmonary function and morphology of C57/BL6N mice. Forty male C57/BL6N mice were randomly divided into two groups: normal control group (20 mice) and cyanate group (20 mice). Mice were exposed to 100 mmol/L cyanate feeding for 4 weeks, and pulmonary Raw (Resistance in Air Way) was measured at the beginning and end of the experiment. The mice were sacrificed at the end of the fourth week of the experiment, and the lung tissues were collected for pathological observation and molecular detection of E-Cadherin and Fibronectin. Well-growing A549 cells in logarithmic growth phase were treated with cyanate at the concentrations of 0, 0.25, 0.5 and 1 mmol/L for 24 h, and the cell viability was detected by CCK8 method; reactive oxygen species ROS fluorescent probe (DCFH-DA) was used to detect the changes of ROS levels, and expressions of E-Cadherin and Fibronectin in cells and pulmonary tissues were detected by Western blot. At the beginning of the experiment, the pulmonary airway resistance values of the mice in the normal control group and the cyanate group were (1.82±0.76)cmH2O/(L·s) and (1.85±0.78)cmH2O/(L·s), respectively, with no significant difference. Four weeks later, the pulmonary airway resistance value of mice in the cyanate group was increased to (4.86±0.87)cmH2O/(L·s) (P＜0.01). The HE staining showed that, compared with the normal control group, the injured alveolar structure, the thickened tracheal wall and the significantly proliferated pulmonary interstitial tissue were observed in the cyanate group. The Masson staining showed that elastic fibers were deposited around the trachea of mice in the cyanate group. The results of CCK8 assay for the viability of A549 cells showed that 0.5 mmol/L cyanate exposure could reduce the viability (P＜0.01). The immunofluorescence staining showed that cyanate could increase ROS level in A549 cells by producing green fluorescence in a concentration-dependent manner. The results of Western blotting showed that 0.5 mmol/L of cyanate treatment on A549 cells could reduce the expression of E-Cadherin (P＜0.01) with increasing concentration of cyanate. The expression level of Fibronectin in A549 cells was increased with the increasing cyanate concentration, and there was a significant difference (P＜0.01) on 1 mmol/L cyanate. Western blot results of lung showed the decreasing expression of E-Cadherin (P＜0.01) and increasing expression of Fibronectin (P＜0.01) in cyanate mice. Pathological concentrations of cyanate can induce the proliferation of pulmonary interstitial tissue, fibrous deposition, and increased pulmonary airway resistance in mice, which may be related to damaged pulmonary epithelial cell viability, enhanced ROS production, and induced pathologic changes of extracellular matrix by cyanate.

Abstract 031: Mutagenesis Of The Cleavage Site Of (pro)renin Receptor Abrogates Aldosterone-salt-induced Hypertension And Renal Injury In Mice

Soluble (pro)renin receptor (sPRR), the extracellular domain of (pro)renin receptor (PRR), is primarily generated by site-1 protease (S1P) and furin. It has been reported that sPRR functions as an important regulator of intrarenal renin contributing to angiotensin II (Ang II)-induced hypertension. Relatively, less is known for the function of sPRR in Ang II-independent hypertension such as mineralocorticoid excess. In the present study, we employed a novel mouse model with mutagenesis of the cleavage site in PRR (termed as Mutant mice) to examine the phenotype during aldosterone (Aldo)-salt treatment. The hypertensive response of Mutant mice to Aldo-salt treatment was blunted (day 12, delta MAP: WT/Aldo-salt 22.4±4.1 mm Hg vs. Mutant/Aldo-salt 11.7±2.5 mm Hg, n=6, p&lt;0.05), in parallel with attenuated response of plasma volume expansion and sodium-water retention. Renal cortical and inner medullary mRNA and protein expression of α-, β-, and γ-subunits of epithelial sodium channel (ENaC) were all increased in WT mice after Aldo-salt treatment but only the increase of α-ENaC in inner medulla was blunted in Mutant mice. The plasma sPRR was increased 1.5-fold after Aldo-salt treatment, which was reduced by 57% in Mutant mice. Moreover, Aldo-salt-induced hypertrophy in the heart and kidney, which were improved in Mutant mice (Heart Weight/Body Weight: WT/Aldo-salt 5.8±0.4 mg/g vs. Mutant/Aldo-salt 4.2±0.3 mg/g; Kidney Weight/Body Weight: WT/Aldo-salt 9.9±0.4 mg/g vs. Mutant/Aldo-salt 7.3±0.4 mg/g, n=6, p&lt;0.01), accompanied with blunted polydipsia and polyuria in Mutant mice. Furthermore, the urinary albumin was increased 8.3-fold after Aldo-salt treatment, which was reduced by 32% in Mutant mice. The mRNA levels of Fibronectin, α-SMA, IL-6, MCP-1 and TGF-β were increased in Aldo-salt/WT mice, which were prevented almost 60% in Aldo-salt/Mutant mice. Together, these results represent strong evidence favoring endogenous sPRR as a mediator of Aldo-salt-induced hypertension and renal injury. Here, we firstly report that sPRR-dependent pathway medicates the Na + -retaining action of Aldo in the distal nephron, which opens up a new area for a better understanding of the molecular basis of renal handling of Na + balance and blood pressure.

Correlation of liver and kidney indicators with foetal vital organ function

This study aimed to investigate the correlation between indicators of liver and kidney function and foetal vital organ function. One hundred and eighty-five pregnant women who underwent cordocentesis and whose foetuses were diagnosed with abnormal foetal organ function were enrolled. The indicators of liver and kidney function were compared between foetuses with abnormal vital organ function and healthy foetuses. There was a significant difference between foetuses with and those without normal cardiovascular systems in terms of total protein, albumin, total bile acid, and creatinine levels (P < .05). A significant difference in aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels was observed in foetuses with and those without normal foetal urinary systems (P < .05). A difference between foetuses with normal and those without normal musculoskeletal systems was noted when comparing LDH levels. Further, there was a significant difference in gestational age and AST, alanine aminotransferase, albumin, total bilirubin, alkaline phosphatase, LDH, adenosine dehydrogenase, fibronectin, and creatinine levels between foetuses with normal versus abnormal blood systems (P < .05). Thus, hepatic and renal function indicators may be associated with abnormal foetal vital organ function. Impact statement What is already known on this subject? Foetal cardiac function is currently evaluated using colour Doppler ultrasound and magnetic resonance imaging in clinical practice, but there are few predictive indicators of the function of other vital organs. It is difficult to determine whether children have abnormalities in the urinary system, digestive system, nervous system, or other vital organs. What do the results of this study add? In this study, it was found that total protein, albumin, total bile acid, creatinine, aspartate aminotransferase, lactate dehydrogenase, fibronectin, alanine aminotransferase, total bilirubin, alkaline phosphatase, adenosine dehydrogenase, and other liver and kidney function indicators may be associated with foetal vital organ dysfunction. However, the forecast range of specific indicators must be further improved upon. What are the implications of these findings for clinical practice and/or further research? This study provides an additional reference for predicting foetal cardiac function.

The Histone Demethylase Inhibitor GSK-J4 Is a Therapeutic Target for the Kidney Fibrosis of Diabetic Kidney Disease via DKK1 Modulation

Diabetic kidney disease (DKD) can cause inflammation and fibrosis, in addition to being the main complication of diabetes. Among many factors, epigenetic alterations in aberrant histone modifications play a key role in causing DKD. In this study, the mechanism of GSK-J4, a histone demethylase KDM6A inhibitor, was evaluated in streptozotocin-induced diabetic mice. It was confirmed that GSK-J4, via dickkopf-1 (DKK1) modulation, could significantly reduce proteinuria and glomerulosclerosis in diabetic mice. The mRNA accumulation levels of DKK1, TGF-β1, fibronectin, and collagen IV were significantly elevated in diabetic mice. In contrast, the mRNA accumulations of those genes were significantly reduced in diabetic mice treated with GSK-J4 compared to those in diabetic mice, relatively speaking. The protein accumulation levels of fibronectin and collagen IV were significantly elevated in diabetic mice. Furthermore, GSK-J4 attenuated the high glucose-induced expression of profibrotic factors in mesangial cells via DKK1. In conclusion, our study provides a novel strategy to eliminate fibrosis in the kidneys of DKD mice. Using GSK-J4 reduces DKK1 expression, thereby ameliorating renal insufficiency, glomerulosclerosis morphological abnormalities, inflammation, and fibrosis in diabetic mice.

FUT8 modulates galectin-3 expression to regulate TGF-β1-mediated fibrosis of lung fibroblasts

To investigate the regulatory role of α-1, 6-fucosyltransferase (FUT8) in TGF-β1-induced proliferation, migration and fibrosis of human embryonic lung fibroblasts (MRC-5 cells) and explore the underlying molecular mechanism. C57/BL6 mice were randomized into 4 groups for treatment with saline (control group), bleomycin, bleomycin+sh-NC or bleomycin+sh-FUT8, and pulmonary fibrosis was observed using Masson staining.MRC-5 cells were transfected with si-NC, FUT8 siRNA (si-FUT8), or both si-FUT8 and a galectin-3(Gal-3) overexpression plasmid (pcDNA3.1-Gal) prior to TGF-β1 treatment, and the changes in cell proliferation and migration were assessed using CCK-8 assay, BrdU assay, and wound healing assay; the changes in the expression levels of α-SMA, collagen I (COLIA1) and extracellular matrix fibronectin (FN) were detected with real-time quantitative PCR (RT-qPCR) and Western blotting.The interaction of FUT8 and Gal-3 was tested using coimmunoprecipitation (Co-IP) assay, and the effect of FUT8 silencing on Gal-3 and FAK/Akt signaling pathways was analyzed. FUT8 knockdown significantly reduced bleomycin-induced extracellular collagen deposition in the lung tissues of the mice.Silencing FUT8 obviously inhibited cell proliferation (P < 0.05) and migration mediated by TGF-β1.FUT8 knockdown down-regulated the mRNA and protein levels of α-SMA, COLIA1 and FN (P < 0.05) in the cells.Coimmunoprecipitation analysis showed that FUT8 interacted with Gal-3.Silencing FUT8 significantly down-regulated Gal-3 expression and inhibited the activation of the FAK/Akt signaling pathway (P < 0.05).Overexpression of Gal-3 obviously reversed the effects of FUT8 silencing on cell proliferation, migration and fibrosis (P < 0.05). FUT8 regulates TGF-β1-induced proliferation, migration and fibrosis of MRC-5 cells by modulating Gal-3 expression, in which the FAK/Akt pathway may play a role.

Gambogenic acid alleviates kidney fibrosis via epigenetic inhibition of EZH2 to regulate Smad7-dependent mechanism

BackgroundEpigenetics regulating gene expression plays important role in kidney fibrosis. Natural products originating from diverse sources including plants and microorganisms are capable to influence epigenetic modifications. Gambogenic acid (GNA) is a caged xanthone extracted from gamboge resin, exudation of Garcinia hanburyi Hook.f., and the effect of GNA on kidney fibrosis with its underlying mechanism on epigenetics remains unknown. PurposeThis study aimed to explore the role of GNA against kidney fibrogenesis by histone methylation mediating gene expression. MethodsTwo experimental mice of unilateral ureteral obstruction (UUO) and folic acid (FA) were given two dosages of GNA (3 and 6 mg/kg/d). TGF-β1 was used to stimulate mouse tubular epithelial (TCMK-1) cells and siRNAs were transfected to verify the underlying mechanisms of GNA. Histological changes were evaluated by HE, MASSON stainings, immunohistochemistry and immunofluorescence. Western blot and qPCR were used to measure protein/gene transcription levels. ResultsGNA dose-dependently alleviated UUO-induced kidney fibrosis and FA-induced kidney early fibrosis, indicated by the pathology and fibrotic factor changes (α-SMA, collagen I, collagen VI, and fibronectin). Mechanically, GNA reduced enhancer of zeste homolog 2 (EZH2) and H3K27me3, promoted Smad7 transcription, and inhibited TGF-β/Smad3 fibrotic signaling in injured kidneys. Moreover, with TGF-β1-induced EZH2 increasing, GNA suppressed α-SMA, fibronectin and collagen levels in tubular epithelial TCMK-1 cells. Although partially decreasing EZH2, GNA did not influence fibrotic signaling in Smad7 siRNA-transfected TCMK-1 cells. ConclusionEpigenetic inhibition of EZH2 by GNA ameliorated kidney fibrogenesis via regulating Smad7-meidated TGF-β/Smad3 signaling.

Explainable Artificial Intelligence Helps in Understanding the Effect of Fibronectin on Survival of Sepsis.

Fibronectin (FN) plays an essential role in the host’s response to infection. In previous studies, a significant decrease in the FN level was observed in sepsis; however, it has not been clearly elucidated how this parameter affects the patient’s survival. To better understand the relationship between FN and survival, we utilized innovative approaches from the field of explainable machine learning, including local explanations (Break Down, Shapley Additive Values, Ceteris Paribus), to understand the contribution of FN to predicting individual patient survival. The methodology provides new opportunities to personalize informative predictions for patients. The results showed that the most important indicators for predicting survival in sepsis were INR, FN, age, and the APACHE II score. ROC curve analysis showed that the model’s successful classification rate was 0.92, its sensitivity was 0.92, its positive predictive value was 0.76, and its accuracy was 0.79. To illustrate these possibilities, we have developed and shared a web-based risk calculator for exploring individual patient risk. The web application can be continuously updated with new data in order to further improve the model.

Effects of Maternal Nicotine Exposure on Fibronectin Expression in the Kidney of Mouse Newborns in Embryonic and Milking Period

Background: Tobacco use in various forms, including hookah, has increased in recent years, especially among young people, who are the group of reproductive age. In the present study, the effect of nicotine on fibronectin expression as a component of basement membrane and extracellular matrix was evaluated in the kidney tissue of neonates whose mothers were exposed to cigarette smoke during pregnancy and lactation period. Methods: Fibronectin expression on days 1, 7, 14, and 21 after delivery was evaluated in the kidneys of Balb/C mice neonates whose mothers were exposed to cigarette smoking during pregnancy and lactation periods. Immunohistochemical and real-time PCR evaluations were performed, and a comparison was made with the control groups. In the experimental groups, nicotine dissolved in saline was injected subcutaneously at a dose of 2 mg/kg daily until the desired day. Results: Our results demonstrated that the expression level of fibronectin increased in nicotine-administrated newborns compared to the healthy controls on days 1 (P = 0.043) and 7 (P = 0.008). The intensity of color reaction on days 1 and 7 was significantly higher in the main kidney structures, including glomeruli and proximal and distal convoluted tubules, in the experimental group than in the control group. The maximum fibronectin expression level was observed on day 7 in the experimental group in comparison with the control group. Conclusions: Nicotine may decrease glomerular filtration rate by increasing fibronectin expression in the basement membrane and extracellular matrix, thereby justifying renal failure in infants exposed to nicotine during embryonic and lactation periods.

Experimental Study on Danggui Shaoyao San Improving Renal Fibrosis by Promoting Autophagy.

Renal fibrosis could lead to chronic kidney disease (CKD) developing into the end-stage with its pathological manifestation is the deposition of extracellular matrix (ECM). Danggui Shaoyao San (DSS) is one of the widely used herbal formulas in ancient China, which has been proven to have efficacy in the treatment of CKD. The experiment employed TGF-β1 to stimulate the NRK-52E cells to establish a renal fibrosis model. With rapamycin (RAPA) used as the positive control, we detected the expression of fibronectin (FN), caspase-3, and autophagy-related proteins in the NRK-52E cells treated with DSS by Western blot and immunofluorescence assay. In order to further verify autophagy-promoting effects of DSS, we adopted 3-MA to inhibit autophagy. The experiment has found that DSS can lower the protein levels of FN and caspase-3 in the NRK-52E cells induced by TGF-β1. After TGF-β1 stimulation, the expression of LC3 II/I and Beclin 1 has decreased, and the protein levels of mTOR and p62 have increased. Consistent with rapamycin, DSS has significantly reduced these effects of TGF-β1. It has also been found that DSS can increase the expression of LC3 II/I and Beclin 1 proteins and can reduce the level of mTOR in cells treated with 3-MA, suggesting that DSS can promote autophagy. In conclusion, DSS has been proved to reduce the apoptosis and fibrosis of NRK-52E cells induced by TGF-β1, which may be achieved by promoting autophagy.

SLC35A2 Deficiency Promotes an Epithelial-to-Mesenchymal Transition-like Phenotype in Madin-Darby Canine Kidney Cells.

In mammalian cells, SLC35A2 delivers UDP–galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin–Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell–cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.

TRPM7 restrains plasmin activity and promotes transforming growth factor-β1 signaling in primary human lung fibroblasts

Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-β1 (TGF-β1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-β1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-β1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-β1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-β1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-β1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-β1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.

Fibronectin: structure, functions, clinical significance (review)

Plasma fibronectin is a high molecular weight adhesive glycoprotein. There are two types of fibronectin: plasma (soluble) and cellular derived (insoluble). Electron microscopy revealed two types of structural organization of fibronectin: compact and expanded. In solution, fibronectin has a compact conformation, and after binding to certain substrates (collagen, fibrin, heparin), it is expanded. Plasma fibronectin is one of the main opsonins of blood plasma in relation to the “targets” of phagocytosis of a predominantly non-bacterial nature, as well as to some types of bacteria. For the treatment of septic processes, as well as respiratory distress syndrome of adults with severe fibronectin deficiency, plasma cryoprecipitate is used – a donor plasma preparation containing a large amount of plasma fibronectin (more than 2 mg/ml). It was proposed to replenish the level of fibronectin in patients with sepsis and other conditions that cause plasma fibronectin deficiency with the help of donor freshly frozen plasma. Transfusion of large volumes of freshly frozen plasma (up to 1000–1500 ml) to patients effectively eliminates the deficiency of plasma fibronectin. The concentration of plasma fibronectin in the blood significantly decreases after the addition of severe infectious processes to hematological diseases, as well as acute DIC syndrome. Extracorporeal methods of blood purification – selective plasmapheresis – have been developed to correct immunocomplex and fibronectin-complex pathology. Two variants of selective plasmapheresis have been proposed: the method of heparinocryoprecipitation of plasma proteins and the method of heparinocryofractionation. In 1987, a plasma heparin precipitate was proposed as a source of fibronectin for the treatment of patients with trophic skin lesions. In 1992, a new method was proposed for obtaining blood preparations with a high concentration of plasma fibronectin from patients themselves (heparin cryofractionation). Autofibronectin preparations obtained by such methods are effective in the local treatment of trophic ulcers in 90–93% of cases. The proposed drugs are safe against infection of patients with infectious diseases transmitted through the blood.

TGF-β1 receptor blockade attenuates unilateral ureteral obstruction-induced renal fibrosis in C57BL/6 mice through attenuating Smad and MAPK pathways.

Renal fibrosis is characterized by accumulation of extracellular matrix components and collagen deposition. TGF-β1 acts as a master switch promoting renal fibrosis through Smad dependent and/or Smad independent pathways. Thirty-five male C57BL/6 mice were divided into five groups of seven each; sham, unilateral ureteral obstruction (UUO), UUO+galunisertib (150 and 300mg/kg/day), galunisertib (300mg/kg/day). The UUO markedly induced renal fibrosis and injury as indicated by renal functional loss, increased levels of collagen Iα1, fibronectin and α-SMA; it also activated both the Smad 2/3 and MAPKs pathways as indicated by increased levels of TGF-β1, p-Smad 2, p-Smad 3, p-p38, p-JNK and p-ERK. These UUO-induced changes were markedly attenuated by oral administration of galunisertib, the TGFβRI small molecule inhibitor. In conclusion, we demonstrated that TGF-β1 receptor blockade can prevent UUO-induced renal fibrosis through indirect modulation of Smad and MAPKs signaling pathways and may be useful as a therapeutic agent in treatment and/or prevention of renal fibrosis.

Abstract 6284: Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells

Abstract Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). We reported miR-29b suppressed MMT of human peritoneal mesothelial cell (HPMC). Methods: MiR-29b mimics and negative control miRNA (NC) were transfected to human bone marrow derived MSC by lipofection method. Exosomes were purified from the culture supernatants of the MSC with ultracentrifugation, and added to HPMC. Proliferation and migration of HPMC were examined with MTT assay and transwell assay, respectively. MMT of HPMC induced by 10 ng/ml TGF-β, and evaluated with immunofluorescence staining. Results: MSC transfected with miR-29b mimics produced significantly increased amounts of miR-29b-containing exosomes (p&amp;lt;0.0001, n=3) as compared with negative control. After 24h-culture with PKH-26 stained exosomes, HPMC actively took the exosomes. After 48 hr-culture with TGF-β, HPMC apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMC was decreased while vimentin and Fibronectin tended to be upregulated by TGF-β. However, exosomes from miR-29b transfected MSC significantly suppressed the morphological changes and reduced the expression levels of vimentin and Fibronectin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMC. The exosome inhibited the proliferation and migration of HPMC by 31±14% (p&amp;lt;0.01, n=5) and 67±9% (p&amp;lt;0.01, n=3), and inhibited the adhesion of NUGC by 90±3% (p&amp;lt;0.001, n=5) as compared with NC exosome. Conclusion: Exosomes from MSC transfected with miR-29b mimic efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Joji Kitayama. Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6284.

Abstract 5540: Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer

Abstract Androgen receptor (AR)-targeted therapies are routinely used to treat prostate cancer, including the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these therapies are initially effective, a significant proportion of patients exhibit resistance to treatment, therefore developing castration-resistant prostate cancer (CRPC). While immune checkpoint blockade therapies have led to remarkable clinical responses in patients with several tumor types, CRPC remains resistant to immunotherapy. The characterization of tumor resistance mechanisms has led to the identification of various tumor variants that could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity. Our laboratory has previously shown that phenotypic plasticity is a driver of resistance to immunotherapy. Here we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways these mechanisms can be mitigated. Models of resistance to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR) were derived from the androgen sensitive LNCAP prostate cancer cell line. Resistant and parental LNCAP cells were comparatively evaluated for phenotypic features via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq analysis. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR and LNCAP parental cells were also grown in vivo in NSG MHCI/II-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets. Our results demonstrated that acquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including increased levels of vimentin and fibronectin, and the loss of epithelial E-cadherin. The susceptibility of LNCAP-Enza-R and LNCAP-AbiR cells to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC) were significantly reduced (up to 90%), compared with parental cells. RNAseq analysis identified several upregulated gene and gene pathways, including estrogen receptor 1 (ESR1), which was particularly overexpressed in LNCAP-EnzaR cells. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets. Our data indicates that acquisition of resistance to AR inhibition is associated with significant reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity features. Future studies plan to investigate approaches that revert tumor plasticity, including blockade of increased markers such as ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC. Citation Format: Madeline Dahut, Kristen Fousek, Lucas Horn, Haiyan Qin, Jeffrey Schlom, Claudia Palena. Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5540.

Abstract 3834: Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma

Abstract Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) have a dismal survival rate. The effects of the extracellular matrix (ECM) on cancer progression have been long studied, but the roles of specific integrins in the process of HNSCC metastasis remain to be dissected. This study aims to determine how HNSCC cells affect the production of laminin-binding integrins, and how these integrins participate in the ECM interactions necessary for a metastatic phenotype. Experimental design: Our laboratory has produced syngeneic mouse SCC cell lines, P029 and A223, derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. In syngeneic recipients, SCCs derived from P029 cells transplanted to the flank mouse skin produced spontaneous metastases to the lung while SCCs derived from A223 cells did not form metastases. Having the same genotype, these cell lines serve as models to examine cancer cell interactions with the ECM and resulting effects on invasion and metastasis. Bulk RNA sequencing (RNAseq) was performed to compare cultured metastatic P029 cells versus non-metastatic A223 cells and identify differentially expressed genes that regulate SCC cells and ECM interactions. Immunoassays and functional invasion assays were performed to evaluate ECM-cancer cell signaling and influence on cancer cell invasion in these two cell lines. Results: RNAseq analysis revealed that, relative to A223 cells, P029 cells have increased levels of integrins, the main mechanoreceptors for numerous ECM ligands and matrix proteins. Laminin-binding integrins, including integrins α4 and β6, were greater at the RNA and protein levels. Additionally, P029 cells expressed higher levels of fibronectin and laminin coding genes compared to non-metastatic A223 cells. Correlatively, high expression of these matrix proteins is associated with worse patient survival. Additionally, P029 SCC cells displayed differential gene expression of tight and apical junctions, PI3K signaling components, and regulators of cytoskeletal remodeling. Furthermore, ELISA and western blot analysis revealed that P029 has aberrant TGFβ-Smad signaling as indicated by the elevated release of TGFβ-1 protein and higher levels of phosphorylated Smad2 and Smad3 relative to non-metastatic A223. Treating P029 cells with TGFβ-1 significantly increased their motility and invasion. Conversely, migration and motility of P029 cells were radically reduced by the TGFβ inhibitor galunisertib. Additionally, the mouse cytokine array revealed that mouse plasma bearing P029 tumors had a greater circulation of CXCL16, MMP-9, proliferin, and serpin E1. These proteins are associated with ECM remodeling and metastasis in HNSCC. Conclusions: SCC cells with metastatic properties upregulate integrins which bind to non-collagen matrix proteins and have elevated invasive and migratory capacity contributed from activated TGFβ signaling. Citation Format: John Aleman, Khoa A. Nguyen, Yao Ke, Christian D. Young, Xiao-Jing Wang. Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3834.