Fibronectin Levels Research Articles

#5333 IMPROVING AUTOPHAGY FLUX BY TFEB ACTIVATION WITH CERIA NANOPARTICLES ATTENUATED CHRONIC KIDNEY INJURY IN CELLULAR AND ANIMAL MODELS OF FABRY DISEASE

Abstract Background and Aims Fabry disease (FD) is a lysosome storage disease (LSD) characterized by significantly reduced intracellular autophagy function. This contributes to the progression of intracellular pathologic signaling and can lead to organ injury. Phospholipid–polyethyleneglycol-capped Ceria-Zirconia antioxidant nanoparticles (PEG-CZNPs) have been reported to enhance autophagy flux. We accessed the action mechanisms of PEG-CZNPs in autophagy regulation and checked the effect on chronic kidney injury in cellular and animal models of FD. Method PEG-CZNPs were synthesized using a non-hydrolytic sol-gel reaction method. HK-2 cells were transfected with α-galactosidase A (α-GLA) shRNA for permanent cellular model of FD. For in-vivo study 4-week-old male B6;129-Glatm1Kul/J mice were treated for 48 weeks with 10mg/kg of PEG-CZNPs twice per week via intraperitoneal injection. PCR, immunoblotting, immunoflouresce assay, electron microscopy analysis, ICP-MS, biochemical and histological analysis were done Results TFEB tanslocated to the nucleus by treatment with PEG-CZNPs. Autophagy flux was evaluated with chloroquine. Autophagy flux was enhanced by PEG-CZNPs treatment. To show whether TFEB plays the important role in autophagy flux, we transfected HK-2 cells with siTFEB. Autophagy flux significantly decreased after knockdown of TFEB with PEG-CZNPs treatment. We next assessed upper signaling pathway of TFEB by PEG-CZNPs. TFEB dephosphorylation was independent of both mTOR and ERK but GSK3ß signaling pathway showed massive impact on TFEB dephosphoryation by PEG-CZNPs. PEG-CZNPs decrease intracellular globotriaosyceramide (Gb3) accumulation and decreased the levels of Collagen type IV, αSMA and MMP9 expression in cellular model of FD. Gb3 levels were significantly reduced in the kidney tissues and the levels of Fibronectin, Collagen type 4 and αSMA was decreased by PEG-CZNPs in animal model of FD. Conclusion These results suggested PEG-CZNPs promote autophagy flux through GSK3ß -TFEB signaling pathways, showed the beneficial effect on renal fibrosis in cellular and animal models of FD. It thus provided a new insights of the potential therapeutics on FD.

#2935 PHARMACOLOGICAL BLOCKADE OF NMDA RECEPTOR DECREASES KIDNEY INJURY INDUCED BY LIPOTOXICITY IN VITRO AND IN VIVO

Abstract Background and Aims Obesity and hyperlipidemia are well established risks factors for kidney injury that can lead to chronic kidney disease (CKD). N-methyl-D-aspartate receptors (NMDARs) are expressed in the kidney and they are involved in the pathology of different renal diseases. The main objective of this study was to establish the role of the NMDAR in the lipotoxicity-induced kidney injury. Method In vivo, we used C57BL/6J mice fed a normal diet (ND) and high fat diet (HFD), as well as mice co-treated with HFD and NMDAR antagonists (HFD+memantine and HFD+MK-801). In vitro, we used renal proximal tubular epithelial cells (HK-2) treated with free fatty acids (FFAs) and a combination of FFAs and NMDAR antagonists. Lipid accumulation was analysed using Oil-Red staining. Expression of NMDAR1 and inflammation, fibrosis and oxidative stress markers were assessed by qPCR and western blot. Results After 10 weeks of diet, mice fed a HFD showed elevated levels of lipids in serum and kidney, compared with the ND. HFD induced an increase of expression of renal NMDAR mRNA and protein, as well as the expression of IL6, ICAM1, MCP1, TNFα and IL1β mRNA. Furthermore, HFD induced an increase of αSMA, vimentin, TGFβ1, fibronectin, OPN and NGAL levels in the kidney. Co-treatment with NMDAR antagonists significantly decreased levels of inflammatory markers, as well as the renal expression of αSMA, OPN and NGAL, but it did not have any effect on the hyperlipidemia induced by HFD. In vitro, treatment of HK-2 with different concentrations of FFAs induced accumulation of lipids in the cell, as well as the increase of the expression of NMDAR1 and markers of inflammation and oxidative stress. Co-treatment with NMDAR antagonists reversed changes induced by FFAs in HK-2 cells. The results were confirmed at the protein level. Conclusion Our results indicate that the NMDAR could be a new therapeutic target in the lipotoxicity-induced kidney injury.

LGALS1 regulates cell adhesion to promote the progression of ovarian cancer.

The present study aimed to explore the significance and molecular mechanisms of galectin-1 (LGALS1) in ovarian cancer (OC). Using the Gene Expression Omnibus database and The Cancer Genome Atlas database, the results of the present study demonstrated that LGALS1 mRNA expression was markedly increased in OC and associated with advanced tumor, lymphatic metastasis and residual lesions. In Kaplan-Meier analysis, patients who expressed LGALS1 highly had a poor prognosis. Furthermore, using The Cancer Genome Atlas database, differentially expressed genes that are potentially regulated by LGALS1 in OC were determined. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis were used to build a biological network of upregulated differentially expressed genes. The results of the enrichment analysis revealed that the upregulated differentially expressed genes were primarily associated with 'ECM-receptor interaction', 'cell-matrix adhesion' and 'focal adhesion', which are closely associated with the metastasis of cancer cells. Subsequently, cell adhesion was selected for further analysis. The results demonstrated that LGALS1 was co-expressed with the candidate genes. Subsequently, the elevated expression levels of candidate genes were verified in OC tissues, and survival analysis indicated that high expression of candidate genes was associated with shortened overall survival of patients with OC. In the present study, OC samples were also collected to verify the high protein expression levels of LGALS1 and fibronectin 1. The results of the present study highlighted that LGALS1 may regulate cell adhesion and participate in the development of OC. Therefore, LGALS1 exhibits potential as a therapeutic target in OC.

Long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense RNA 1 regulates myocardial fibrosis in diabetes through the Hippo-Yes-associated protein signaling pathway.

Fibrosis is the principle reason for heart failure in diabetes. Regarding the involvement of long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) in diabetic myocardial fibrosis, we explored its specific mechanism. Human cardiac fibroblasts (HCF) were treated with high glucose (HG) and manipulated with plasmid cloning deoxyribonucleic acid 3.1-ZEB1-AS1/microribonucleic acid (miR)-181c-5p mimic/short hairpin RNA specific to sirtuin 1 (sh-SIRT1). ZEB1-AS1, miR-181c-5p expression patterns, cell viability, collagen I and III, α-smooth muscle actin (α-SMA), fibronectin levels and cell migration were assessed by reverse transcription quantitative polymerase chain reaction, cell counting kit-8, western blot and scratch tests. Nuclear/cytosol fractionation assay verified ZEB1-AS1 subcellular localization. The binding sites between ZEB1-AS1 and miR-181c-5p, and between miR-181c-5p and SIRT1 were predicted and verified by Starbase and dual-luciferase assays. The binding of SIRT1 to Yes-associated protein (YAP) and YAP acetylation levels were detected by co-immunoprecipitation. Diabetic mouse models were established. SIRT1, collagen I, collagen III, α-SMA and fibronectin levels, mouse myocardium morphology and collagen deposition were determined by western blot, and hematoxylin-eosin and Masson trichrome staining. Zinc finger E-box binding homeobox 1 antisense 1 was repressed in HG-induced HCFs. ZEB1-AS1 overexpression inhibited HG-induced HCF excessive proliferation, migration and fibrosis, and diminished collagen I, collagen III, α-SMA and fibronectin protein levels in cells. miR-181c-5p had targeted binding sites with ZEB1-AS1 and SIRT1. SIRT1 silencing/miR-181c-5p overexpression abrogated ZEB1-AS1-inhibited HG-induced HCF proliferation, migration and fibrosis. ZEB1-AS1 suppressed HG-induced HCF fibrosis through SIRT1-mediated YAP deacetylation. ZEB1-AS1 and SIRT1 were repressed in diabetic mice, and miR-181c-5p was promoted. ZEB1-AS1 overexpression improved myocardial fibrosis in diabetic mice, and reduced collagen I, collagen III, α-SMA and fibronectin protein levels in myocardial tissues. Long non-coding ribonucleic acid ZEB1-AS1 alleviated myocardial fibrosis through the miR-181c-5p-SIRT1-YAP axis in diabetic mice.

Vincamine Ameliorates Epithelial-Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis in Rats; Targeting TGF-β/MAPK/Snai1 Pathway.

Idiopathic pulmonary fibrosis is a progressive, irreversible lung disease that leads to respiratory failure and death. Vincamine is an indole alkaloid obtained from the leaves of Vinca minor and acts as a vasodilator. The present study aims to investigate the protective activity of vincamine against EMT in bleomycin (BLM)-induced pulmonary fibrosis via assessing the apoptotic and TGF-β1/p38 MAPK/ERK1/2 signaling pathways. In bronchoalveolar lavage fluid, protein content, total cell count, and LDH activity were evaluated. N-cadherin, fibronectin, collagen, SOD, GPX, and MDA levels were determined in lung tissue using ELISA. Bax, p53, bcl2, TWIST, Snai1, and Slug mRNA levels were examined using qRT-PCR. Western blotting was used to assess the expression of TGF-β1, p38 MAPK, ERK1/2, and cleaved caspase 3 proteins. H & E and Masson's trichrome staining were used to analyze histopathology. In BLM-induced pulmonary fibrosis, vincamine reduced LDH activity, total protein content, and total and differential cell count. SOD and GPX were also increased following vincamine treatment, while MDA levels were decreased. Additionally, vincamine suppressed the expression of p53, Bax, TWIST, Snail, and Slug genes as well as the expression of factors such as TGF-β1, p/t p38 MAPK, p/t ERK1/2, and cleaved caspase 3 proteins, and, at the same time, vincamine increased bcl2 gene expression. Moreover, vincamine restored fibronectin, N-Catherine, and collagen protein elevation due to BLM-induced lung fibrosis. In addition, the histopathological examination of lung tissues revealed that vincamine attenuated the fibrotic and inflammatory conditions. In conclusion, vincamine suppressed bleomycin-induced EMT by attenuating TGF-β1/p38 MAPK/ERK1/2/TWIST/Snai1/Slug/fibronectin/N-cadherin pathway. Moreover, it exerted anti-apoptotic activity in bleomycin-induced pulmonary fibrosis.

Human umbilical cord mesenchymal stem cell exosomes alleviate acute kidney injury by inhibiting pyroptosis in rats and NRK-52E cells

Human umbilical cord mesenchymal stem cells (hucMSCs) have been shown to improve kidney injury. Exosomes have been indicated to be important mediators of renal protection in MSC therapy. In spite of this, the mechanism remains unclear. Our study investigated how exosomes derived from hucMSCs (hucMSC-Ex) improve acute kidney injury (AKI). Exosomes were extracted by using an ultracentrifugation technique and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Twenty-four male SD rats were randomly divided into four groups: sham group, sham + hucMSC-Ex group, ischemia–reperfusion injury (IRI) group, and IRI + hucMSC-Ex group. In vitro, we treated rat proximal renal tubular epithelial cell line (NRK-52E) with cisplatin to mimic in vivo models of AKI. The NRK-52E cells were treated with or without 160 μg/mL hucMSC-Ex, and 1 µg/mL cisplatin was added after 9 h. Cells were harvested after 24 h. In the IRI group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were increased; renal tubules were dilated, epithelial cells were vacuolated, and collagen fibers were deposited in the renal interstitium. After treatment with cisplatin, the NRK-52E cells displayed pyroptotic morphology characterized by pyroptotic bodies. The protein expression levels of fibronectin, α-smooth muscle actin (α-SMA), vimentin, gasdermin D (GSDMD), caspase-1, interleukin-1 (IL-1β) and NLRP3 in IRI tissues and in cisplatin treatment NRK-52E cells were significantly upregulated. However, after the hucMSC-Ex intervention, kidney injury was effectively improved in vivo and in vitro. The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis.

Crosstalk between Interleukin-1 Receptor-Like 1 and Transforming Growth Factor-β Receptor Signaling Promotes Renal Fibrosis

IL-33, a member of the IL-1 family, acts as an alarmin in immune response. Epithelial-mesenchymal transition and transforming growth factor-β (TGF-β)–induced fibroblast activation are key events in the development of renal interstitial fibrosis. The current study found increased expression of IL-33 and interleukin-1 receptor-like 1 (IL1RL1, alias ST2), the receptor for IL-33, in human fibrotic renal tissues. In addition, IL-33– or ST2-deficient mice showed significantly reduced levels of fibronectin, α-smooth muscle actin, and vimentin, and increased E-cadherin levels. In HK-2 cells, IL-33 promotes the phosphorylation of the TGF-β receptor (TGF-βR), Smad2, and Smad3, and the production of extracellular matrix (ECM), with reduced expression of E-cadherin. Blocking TGF-βR signaling or suppressing ST2 expression impeded Smad2 and Smad3 phosphorylation, thereby reducing ECM production, suggesting that IL-33–induced ECM synthesis requires cooperation between the two pathways. Mechanistically, IL-33 treatment induced a proximate interaction between ST2 and TGF-βRs, activating downstream Smad2 and Smad3 for ECM production in renal epithelial cells. Collectively, this study identified a novel and essential role for IL-33 in promoting TGF-β signaling and ECM production in the development of renal fibrosis. Therefore, targeting IL-33/ST2 signaling may be an effective therapeutic strategy for renal fibrosis.

Enhanced reparatory effect of EI1 on dental pulp via extracellular matrix remodeling by miR-181b-2-3p inhibitor

Background/purposeExtracellular matrix (ECM) is crucial for dental pulp repair. The aim of this paper is to investigate the ECM remodeling effect of miR-181b-2-3p (a microRNA) and to verify the reparatory effect of EI1 (an epigenetic drug) and miR-181b-2-3p inhibitor on dental pulp. Materials and methodsLevels of ECM-related factors in EI1-treated human dental pulp cells (hDPCs) were measured by qRT-PCR and Western blot. The anti-inflammation effect of EI1 was examined in Lipopolysaccharide-stimulated hDPCs. miR-181b-2-3p mimics or inhibitors were transfected into hDPCs and then the cells’ functions were detected. A dual luciferase reporter assay was used to identify the targets of miR-181b-2-3p. Pulpotomy using miR-181b-2-3p antagomirs and EI1 as pulp capping materials was performed in male six-week-old Sprague-Dawley rats. ResultsEI1 upregulated ECM-related genes expression in hDPCs, but failed to upregulate the collagen1A1 (COL1A1) protein level. Pro-inflammatory factors were downregulated by EI1 in Lipopolysaccharide-stimulated hDPCs. Overexpression of miR-181b-2-3p downregulated the expression of transforming growth factor-β2 (TGF-β2) and fibronectin type III domain-containing protein 5 precursor (FNDC5), while the inhibition had the opposite effect. Dual luciferase reporter assays demonstrated that miR-181b-2-3p targets TGF-β2, FNDC5 and integrin alpha 4 protein (ITGA4). Compared to EI1 was used alone, EI1 combined with the inhibitor upregulated the protein levels of COL1A1, fibronectin (FN1) and TGF-β2 in hDPCs, promoted hDPCs migration, and exhibited reparatory effects on inflamed rat pulp tissue. ConclusionmiR-181b-2-3p inhibitor could enhance the reparatory effect of EI1 via ECM remodeling in dental pulp both in vitro and in vivo.

ALDH2 promotes cancer stemness and metastasis in colorectal cancer through activating β-catenin signaling.

Colorectal cancer (CRC) is the primary cause of death from gastrointestinal cancers. Aldehyde dehydrogenase 2 (ALDH2), a crucial mitochondrial enzyme for the oxidative pathway of alcohol metabolism, plays a dual role in cancer progression. In some cancers, it is tumor suppressive; in others, it drives cancer progression. However, whether targeting ALDH2 has any therapeutic implications or prognostic value in CRC is still unclear. Here, we investigated the role of ALDH2 in CRC progression by targeting its enzymatic activity rather than gene expression. We found that inhibiting ALDH2 by CVT-10216 and daidzein significantly decrease migration and stemness properties of both DLD-1 and HCT 116 cells, whereas activating ALDH2 by Alda-1 enhances migration rate. Concomitantly, ALDH2 inhibition by both CVT-10216 and daidzein downregulates the mRNA levels of fibronectin, snail, twist, MMP7, CD44, c-Myc, SOX2, and OCT-4, which are oncogenic in the advanced stage of CRC. Furthermore, Gene Set Enrichment Analysis (GSEA) on ALDH2 co-expressed genes from The Cancer Genome Atlas (TCGA) revealed that MYC target gene sets are upregulated. We found that ALDH2 inhibition decreased the nuclear protein levels of pGSK3β serine 9 and c-Myc. This suggests that ALDH2 probably targets β-catenin signaling in CRC cells. Together, our results demonstrate the prognostic value of ALDH2 in CRC as it regulates both CRC stemness and migration. Our findings also propose that the plant-derived isoflavone daidzein could be a potential chemotherapeutic drug targeting ALDH2 in CRC.

MiR-21-5p promotes sorafenib resistance and hepatocellular carcinoma progression by regulating SIRT7 ubiquitination through USP24

ObjectiveTo validate the mechanism by which miR-21-5p mediates autophagy in drug-resistant cells in hepatocellular carcinoma (HCC), aggravating sorafenib resistance and progression of HCC. MethodsHCC cells were treated with sorafenib to establish sorafenib-resistant cells, and nude mice were subcutaneously injected with hepatoma cells to establish animal models. RT–qPCR was used to determine the level of miR-21-5p, and Western blotting was used to determine the level of related proteins. Cell apoptosis, cell migration, the level of LC3 were accessed. Immunohistochemical staining was used for detection of Ki-67 and LC3. A dual-luciferase reporter assay certified that miR-21-5p targets USP42, and a co-immunoprecipitation assay validated the mutual effect between USP24 and SIRT7. ResultsmiR-21-5p and USP42 were highly expressed in HCC tissue and cells. Inhibition of miR-21-5p or knockdown of USP42 inhibited cell proliferation and cell migration, upregulated the level of E-cadherin, and downregulated the level of vimentin, fibronectin and N-cadherin. Overexpression of miR-21-5p reversed the knockdown of USP42. Inhibition of miR-21-5p downregulated the ubiquitination level of SIRT7, downregulated the levels of LC3II/I ratio and Beclin1, and upregulated the expression of p62. The tumor size in the miR-21-5p inhibitor group was smaller, and Ki-67 and LC3 in tumor tissue were reduced, while the overexpression of USP42 reversed the effect of the miR-21-5p inhibitor. ConclusionmiR-21-5p promotes deterioration and sorafenib resistance in hepatocellular carcinoma by upregulating autophagy levels. Knockdown of miR-21-5p inhibits the development of sorafenib-resistant tumors by USP24-mediated SIRT7 ubiquitination.

Integrin β6 mediates epithelial–mesenchymal transition in diabetic kidney disease

The progression of diabetic kidney disease (DKD) is associated with increased fibronectin (FN) levels in proximal tubular epithelial cells. Bioinformatics analysis showed that integrin β6 and cell adhesion function were significantly changed in the cortices of db/db mice. Remodelling of cell adhesion is one of the core changes during epithelial–mesenchymal transition (EMT) in DKD. Integrin is a family of transmembrane proteins that regulates cell adhesion and migration, and extracellular FN is the major ligand of integrin β6. We found that the expression of integrin β6 was elevated in the proximal tubules of db/db mice and FN-induced renal proximal tubule cells. The levels of EMT were also significantly increased in vivo and in vitro. In addition, FN treatment activated the Fak/Src pathway, increased the expression of p-YAP, and then upregulated the Notch1 pathway in diabetic proximal tubules. Knockdown of integrin β6 or Notch1 reduced the EMT aggravation induced by FN. Furthermore, urinary integrin β6 was significantly increased in DKD patients. Our findings reveal a critical role of integrin β6 in regulating EMT in proximal tubular epithelial cells and identify a novel direction for the detection and treatment of DKD.

Fufang Shenhua tablet inhibits renal fibrosis by inhibiting PI3K/AKT

BackgroundFufang Shenhua tablet (SHT), a traditional Chinese medicine compound, has been utilized in the clinical management of chronic kidney disease (CKD) for a long time. Nevertheless, the fundamental active constituents and potential mechanism of action remain unclear. Thus, the objective of this study was to investigate the renoprotective effect of SHT on residual renal tissue in CKD model rats and to explore its primary efficacious components and their underlying mechanism. MethodsAfter a 12-week period of SHT treatment through gavage in a 5/6 nephrectomized animal model of CKD, we evaluated the body weight, renal function, and renal pathological changes. Furthermore, the expression levels of fibronectin (FN), collagen I (COL-1), α-smooth muscle actin (α-SMA), and vimentin in renal tissues were assessed. In addition, network pharmacology analysis and molecular docking were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways through which SHT could potentially exert its anti-kidney fibrosis effects. Subsequently, these predictions were validated in renal tissues of rats with CKD and in transforming growth factor β1 (TGF-β1)-induced HK-2 cells. ResultsSHT significantly improved renal function and reduced renal pathological damage and fibrosis in CKD model rats. Network pharmacological analysis identified 62 active components in SHT, with quercetin ranked first, and 105 protein targets shared by SHT and CKD. Based on the protein‒protein interaction network (PPI) and the SHT-CKD-pathway network, AKT1, MYC, IL2, and VEGFA were identified as key targets. Furthermore, GO and KEGG pathway enrichment analyses indicated that the renoprotective effect of SHT on CKD was closely associated with the PI3K/AKT signaling pathway. Molecular docking results demonstrated that the main active components of SHT had a strong binding affinity to the hub genes. During experimental validation, SHT hindered the activity of the PI3K/AKT signaling pathway in the renal tissue of CKD model rats. Furthermore, activation of the PI3K/AKT signaling pathway was correlated with a modified fibrotic phenotype in rats with 5/6 nephrectomy-induced CKD and TGF-β1-induced HK-2 cells. Conversely, SHT and quercetin curtailed the activation of the PI3K/AKT signaling pathway and inhibited the formation of renal fibrosis, thus indicating that the PI3K/AKT signaling pathway is the basis of the antifibrotic effects of SHT. Ultimately, administration of the PI3K/AKT agonist 740Y-P counteracted the fibrotic phenotype of TGF-β1-induced HK-2 cells induced by SHT. ConclusionsIn this investigation, we employed a fusion of systems pharmacology and in vivo and in vitro experiments to elucidate the mechanism of SHT's antifibrotic properties via obstruction of the PI3K/AKT signaling pathway. Additionally, we surmised that AKT may be the principal target of SHT for the management of CKD and that quercetin may be its efficacious component. We have thus identified SHT as a promising drug for the amelioration of renal fibrosis and the progression of CKD.

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.

Therapeutic administration of mouse mast cell protease 6 improves functional recovery after traumatic spinal cord injury in mice by promoting remyelination and reducing glial scar formation.

Traumatic spinal cord injury (SCI) most often leads to permanent paralysis due to the inability of axons to regenerate in the adult mammalian central nervous system (CNS). In the past, we have shown that mast cells (MCs) improve the functional outcome after SCI by suppressing scar tissue formation at the lesion site via mouse mast cell protease 6 (mMCP6). In this study, we investigated whether recombinant mMCP6 can be used therapeutically to improve the functional outcome after SCI. Therefore, we applied mMCP6 locally via an intrathecal catheter in the subacute phase after a spinal cord hemisection injury in mice. Our findings showed that hind limb motor function was significantly improved in mice that received recombinant mMCP6 compared with the vehicle-treated group. In contrast to our previous findings in mMCP6 knockout mice, the lesion size and expression levels of the scar components fibronectin, laminin, and axon-growth-inhibitory chondroitin sulfate proteoglycans were not affected by the treatment with recombinant mMCP6. Surprisingly, no difference in infiltration of CD4+ T cells and reactivity of Iba-1+ microglia/macrophages at the lesion site was observed between the mMCP6-treated mice and control mice. Additionally, local protein levels of the pro- and anti-inflammatory mediators IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFNγ, and MCP-1 were comparable between the two treatment groups, indicating that locally applied mMCP6 did not affect inflammatory processes after injury. However, the increase in locomotor performance in mMCP6-treated mice was accompanied by reduced demyelination and astrogliosis in the perilesional area after SCI. Consistently, we found that TNF-α/IL-1β-astrocyte activation was decreased and that oligodendrocyte precursor cell (OPC) differentiation was increased after recombinant mMCP6 treatment in vitro. Mechanistically, this suggests effects of mMCP6 on reducing astrogliosis and improving (re)myelination in the spinal cord after injury. In conclusion, these data show for the first time that recombinant mMCP6 is therapeutically active in enhancing recovery after SCI.

Profiling of idiopathic macular hole vitreous proteome identifies the role of extracellular matrix remodelling, epithelial–mesenchymal transformation and unfolded protein-response pathways

Purpose: To analyze and describe the proteome of the vitreous humour in eyes with idiopathic macular holes. Methods: We performed mass spectrometry (MS)-based label-free quantitative analysis of the vitreous proteome of idiopathic macular hole (IMH) and control donor vitreous. Comparative quantification was performed using SCAFFOLD software which calculated fold changes of differential expression. Bioinformatics analysis was performed using DAVID and STRING software. Results: A total of 448 proteins were identified by LC-MS/MS in IMH and cadaveric eye vitreous samples, of which 199 proteins were common. IMH samples had 189 proteins that were unique and 60 proteins were present only in the control cadaveric vitreous. We found upregulation of several extracellular matrix (ECM) and cytoskeletal proteins, namely collagen alpha-1 (XVIII) chain, N-cadherin, EFEMP1/fibulin-3, basement membrane-specific heparan sulfate proteoglycan core protein, and target of Nesh-3. Several cytoskeleton proteins, namely tubulin, actin, and fibronectin levels, were significantly lower in IMH vitreous, probably reflecting increased ECM degradation. IMH vitreous also had a downregulation of unfolded protein response-mediated-mediated apoptosis proteins, possibly related to a state of increased cell survival and proliferation, along with a remodelling and aberrant production of ECM contents. Conclusion: The pathogenesis of macular holes may involve ECM remodelling, epithelial–mesenchymal transformation, downregulation of apoptosis, protein folding defects, and complement pathway. The vitreo-retinal milieu in macular holes contain molecules related to both ECM degradation and inhibition of the same, thereby maintaining a homeostasis.

N-3 Fatty Acid Supplementation After Burn Supports Wound Healing and Prevents Systemic Inflammation. An Experimental Study in Rats

Objective: Burn may cause hypersensitivity towards infection, systemic inflammatory response syndrome, respiratory distress syndrome and multiple organ failure. Anti-inflammatory and resistance towards infection effects of omega-3 (n-3) fatty acids was shown before. But their effect on systemic inflammation and wound healing after burn was not investigated. 
 Methods: Seventy Wistar albino rats were used in the study. Fourteen rats without burn composed the control group. Cutaneous burn was created on remaining 56 rats which received 50 mg/kg/day intraperitoneal ringer lactat solution in two equal amounts. Twenty-eight rats with burn received injections of n-3 1 mg/kg/day throughout the study. Rats in control group were sacrificed on day 4 and rats in treatment groups were sacrificed on days 4 and 8. Leukocyte and mast cell infiltration, epidermal and collagen thickness measurements were used in histological evaluation. White blood cell, hematocrite, total protein, albumin, fibronectin, TNFα, IL1β and IL6 levels were measured for biochemical evaluations. 
 Results: Elevation of leukocyte and mast cell infiltration was lower and collagen thickness was higher in n-3 receiving group on day 4. There was no difference in leukocyte infiltration on the 8th day whereas mast cell infiltration was lower and collagen thickness was higher in n-3 receiving group. Epidermal thickness was higher in n-3 receiving group on both evaluation days. White blood cell counts were higher in 8th day than 4th day groups. Total protein on 8th, IL-6 on 4th, and albumin levels on both days was lower than control.
 Conclusion: N-3 fatty acids used after burn enhances wound healing and prevents systemic inflammation.

EFFECT OF CARDIOPROTECTIVE THERAPY ON INFLAMMATION AND FIBROSIS FORMATION IN PATIENTS WITH MYOCARDIAL INFARCTION IN COMBINATION WITH TYPE 2 DIABETES MELLITUS

Myocardial infarction, especially ST-elevation myocardial infarction (STEMI), is the leading cause of death worldwide. Rapid detection and treatment of patients with STEMI is essential to minimize heart muscle damage and improve patient outcomes. Type 2 diabetes mellitus (T2DM) is a common metabolic disorder that is associated with various cardiovascular diseases, including STEMI. Patients with T2DM have a higher risk of developing STEMI, and this relationship is explained by factors such as insulin resistance, oxidative stress, and low-grade inflammation. Low-grade inflammation is now considered a characteristic feature of T2DM associated with the development of cardiovascular disease.
 Aim: to evaluate the effect of quercetin and adenosine-containing drug Advocard on inflammation and myocardial remodeling in patients with STEMI and concomitant T2DM.
 Materials and methods. This study involved 97 patients who were diagnosed with STEMI and T2DM who were inpatients at the Ivano-Frankivsk Regional Clinical Cardiology Center. The patients were divided into three separate groups. The first group included 34 patients who received only standard treatment as prescribed by current protocols for STEMI and T2D. The second group consisted of 29 patients who received the same basic therapy, but with the addition of quercetin (4.5 g intravenously for 5 days) to the treatment complex. The third group included 34 patients who received quercetin and Advocard (adenosine -29.25 mg in combination with molsidomine - 0.3 mg and folic acid - 0.45 mg in one half tablet) 2 tablets 3 times a day for 3 weeks against the background of similar basic therapy.
 Results. After analyzing the data of the three groups, it was found that the mean fibronectin level before treatment was 2.92±0.11 ng/ml in group 1, 2.67±0.39 ng/ml in group 2 and 2.68±0.38 ng/ml in group 3 (group 1 vs. group 2 p=0.892, group 1 vs. group 3 p=0.12). After treatment, the average fibronectin level decreased to 1.82±0.35 ng/ml in group 1, 1.11±0.26 ng/ml in group 2, and 0.82±0.28 ng/ml in group 3 (group 1 vs. group 2 p<0.001, group 1 vs. group 3 p<0.001). The difference between pre- and post-treatment levels (Δ fibronectin) was 0.86 ng/ml in group 1, 1.57 ng/ml in group 2, and 2.10 ng/ml in group 3. The mean level of sST2 before treatment was 23.08±1.39 ng/ml in the first group, 22.96±1.13 ng/ml in the second group and 23.11±1.05 ng/ml in the third group (group 1 vs. group 2, p=0.705, group 1 vs. group 3, p=0.916). After treatment, the mean level of sST2 decreased to 15.88±1.76 ng/mL in the first group, 10.06±1.77 ng/mL in the second group, and 7.11±1.65 ng/mL in the third group (group 1 vs. group 2 p<0.001, group 1 vs. group 3 p<0.001). The difference between the pre- and post-treatment levels, or Δ sST2, was 7.21 ng/mL in the first group, 12.90 ng/mL in the second group, and 16.00 ng/mL in the third group.
 Conclusions:
 The use of quercetin and advocard in the pharmacological management of patients with STEMI and T2DM reduces the intensity of inflammation and fibrosis, as evidenced by a decrease in the levels of fibronectin and sST2 in the blood.

Label-free quantitative proteomics reveals aberrant expression levels of LRG, C9, FN, A1AT and AGP1 in the plasma of patients with colorectal cancer

BackgroundColorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Although commercial biomarkers of CRC are currently available, they are still lacking in terms of sensitivity and specificity; thus, searching for reliable blood-based biomarkers are important for the primary screening of CRC.MethodsPlasma samples of patients with non-metastatic (NM) and metastatic (M) CRC and healthy controls were fractionated using MARS-14 immunoaffinity chromatography. The flow-through and elute fractions representing low- and high-abundant proteins, respectively, were analyzed by label-free quantitative proteomics mass spectrometry. The functional analysis of the proteins with greater than 1.5-fold differential expression level between the CRC and the healthy control groups were analyzed for their biological processes and molecular functions. In addition, the levels of plasma proteins showing large alterations in CRC patients were confirmed by immunoblotting using two independent cohorts. Moreover, receiver operating characteristic (ROC) curve analysis was performed for individual and combinations of biomarker candidates so as to evaluate the diagnostic performance of biomarker candidates.ResultsFrom 163 refined identifications, five proteins were up-regulated and two proteins were down-regulated in NM-CRC while eight proteins were up-regulated and three proteins were down-regulated in M-CRC, respectively. Altered plasma proteins in NM-CRC were mainly involved in complement activation, while those in M-CRC were clustered in acute-phase response, complement activation, and inflammatory response. Results from the study- and validation-cohorts indicate that the levels of leucine-rich alpha-2-glycoprotein-1(LRG), complement component C9 (C9), alpha-1-acid glycoprotein 1 (AGP1), and alpha-1-antitrypsin (A1AT) were statistically increased, while fibronectin (FN) level was statistically decreased in CRC patients compared to healthy controls, with most alterations found in a metastatic stage-dependent manner. ROC analysis revealed that FN exhibited the best diagnostic performance to discriminate CRC patients and healthy controls while AGP1 showed the best discrimination between the disease stages in both cohorts. The combined biomarker candidates, FN + A1AT + AGP1, exhibited perfect discriminatory power to discriminate between the CRC population and healthy controls whereas LRG + A1AT + AGP1 was likely to be the best panel to discriminate the metastatic stages in both cohorts.ConclusionsThis study identified and quantified distinct plasma proteome profiles of CRC patients. Selected CRC biomarker candidates including FN, LRG, C9, A1AT, and AGP1 may be further applied for screening larger cohorts including disease groups from other types of cancer or other diseases.

Abstract 5808: Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemo-resistance and fatal tumor recurrence after primary treatment. The metastatic progression is mediated by exfoliation of tumor cells into the peritoneal cavity, the formation of compact OC spheroids, and their implantation onto the overlying peritoneal surface. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components, such as fibronectin (FN), that stiffen the tumor stroma, promote the aberrant activation of integrins, and recruit the adaptor protein integrin-linked kinase (ILK). ILK drives cytoskeletal assembly and modulates key processes, including survival, invasion, and stemness. However, the functional role of ILK in chemoresistance of ovarian cancer stem cells (OCSCs) remains incompletely understood. We tested the hypothesis that the formation of FN-integrin complexes at the cell membrane promotes survival of OCSCs and supports OC spheroid formation by activating ILK and downstream oncogenic signaling. In OCSCs compared with non-CSC, ILK, FN, and integrin β1 mRNA expression levels were increased (P<0.001) and further enriched in OC spheroids compared with monolayers (P<0.01). ILK blockade with the specific inhibitor cpd-22 suppressed spheroid proliferation and tumor initiating capacity in xenograft mouse model. Furthermore, key oncogenic signaling pathways, in particular decreased β-catenin signaling essential to sustaining cancer cell stemness, were altered in OC spheroids treated with cpd-22. Of the genes examined, expression of Wnt receptor Frizzled 7 (Fzd7) was the mostly highly downregulated in OC spheroids treated with cpd-22, indicating a direct correlation between ILK activation and Wnt signaling. By using co-immunoprecipitation and proximity ligation assay, we demonstrated that ILK co-localizes with Fzd7 in OC spheroids. Mechanistically, treatment of OC spheroids with the combination of carboplatin and ILK-Fzd7 blockade decreased β-catenin signaling, inhibited phospho-Akt at Ser473 and increased levels of cleaved-caspase-3 compared to single agent alone, resulting in apoptosis. By regulating β-catenin signaling, Fzd7 expression and ILK activation are essential for OCSCs survival. Targeting Fzd7-ILK clusters may represent a new therapeutic approach to eradicate OCSC and ultimately improve patient outcomes. Citation Format: Rula Atwani, Virginie Lazar, George Earl Sandusky, Salvatore Condello. Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells. [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 5808.

Tenascin-C promotes the proliferation and fibrosis of mesangial cells in diabetic nephropathy through the β-catenin pathway.

To mechanistically assess the involvement of tenascin-C (TNC) in diabetic nephropathy (DN). Renal specimens from DN patients were histopathologically examined, and their TNC expression patterns were evaluated via immunohistochemistry. Additionally, the hereditarily diabetic C57BL/KsJ db/db mice were induced to develop DN via adaptive feeding, and then their renal levels of TNC and β-catenin were assessed via western blotting and immunohistochemistry. Furthermore, the TNC and β-catenin levels in primary rat mesangial cells (RMCs) cultured with high glucose levels were assessed via western blotting. In parallel, RMCs cultured with TNC in the presence or absence of the β-catenin blocker ICG-001 were analyzed for their fibronectin and collagen I levels via immunostaining, and for their fibronectin, α-SMA, vimentin, PDGFR-β, PCNA, and β-catenin levels via western blotting. The TNC levels in the specimens were associated with the pathological classification. In these DN specimens, TNC protein was highly detected in the MCs and slightly in the tubulointerstitium. Renal TNC (P < 0.05) and β-catenin (P < 0.001) were upregulated in db/db vs. db/m mice. High-glucose treatment upregulated TNC (P < 0.01) and β-catenin (P < 0.05) in RMCs. TNC treatment upregulated fibronectin (P < 0.05), α-SMA (P < 0.01), vimentin (P < 0.05), PCNA (P < 0.05), and β-catenin (P < 0.05) in RMCs, as assessed via western blotting. Immunohistochemical analysis confirmed the fibronectin upregulation and showed collagen I upregulation. Western-blot results also showed that levels of fibronectin (P < 0.001), α-SMA (P < 0.01), vimentin (P < 0.001), PCNA (P < 0.05), PDGFR-β (P < 0.05), and β-catenin (P < 0.01) were lower in RMCs co-treated with TNC and ICG-001 than in TNC-treated cells. Immunofluorescence analysis confirmed the decreased fibronectin level and showed that the collagen I level was also decreased by ICG-001. TNC is upregulated in DN and induces MC proliferation and fibrosis through the β-catenin pathway.