Unboxing the network among long non-coding RNAs and TGF-ß signaling in cancer.

Deeper analysis of molecular mechanisms arising in tumor cells is an unmet need to provide new diagnostic and therapeutic strategies to prevent and treat tumors. The transforming growth factor ß (TGF-ß) signaling has been steadily featured in tumor biology and linked to poor prognosis of cancer patients. One pro-tumorigenic mechanism induced by TGF-ß is the epithelial-to-mesenchymal transition (EMT), which can initiate cancer dissemination, enrich the tumor stem cell population, and increase chemoresistance. TGF-ß signals via SMAD proteins, ubiquitin ligases, and protein kinases and modulates the expression of protein-coding and non-coding RNA genes, including those encoding larger than 500 nt transcripts, defined as long non-coding RNAs (lncRNAs). Several reports have shown lncRNAs regulating malignant phenotypes by directly affecting epigenetic processes, transcription, and post-transcriptional regulation. Thus, this review aims to update and summarize the impact of TGF-ß signaling on the expression of lncRNAs and the function of such lncRNAs as regulators of TGF-ß signaling, and how these networks might impact specific hallmarks of cancer.

"Decoding inflammation: glycoprotein a repetition predominant, microRNA-142-3-p, and metastasis associated lung adenocarcinoma transcript 1: as novel inflammatory biomarkers of inflammatory bowel disease".

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic gastrointestinal (GI) condition comprising Crohn's disease (CD) and ulcerative colitis (UC). The pathogenesis involves immune system dysregulation, with increased Th (T helper cell)17 cells and reduced regulatory T cell (Treg) differentiation. Transforming growth factor-ß (TGF-ß) secretion from Tregs helps control inflammation, and its production is regulated by glycoprotein-A repetition predominant (GARP) protein along with non-coding RNAs (ncRNAs) like microRNA(miR)-142-3p and metastasis associated lung adenocarcinoma transcript 1 (MALAT1) long non-coding RNAs (LncRNAs). This study analyzed their expression in IBD. METHODS: Blood samples were collected from 44 IBD patients, and 22 healthy controls (HC). RNA extraction and circular DNA (cDNA) synthesis were performed. Real-time polymerase chain reaction (RT-PCR) measured gene expression of GARP, MALAT1, and miR-142-3p. Correlations and group differences were statistically analyzed. RESULTS: Compared to controls, GARP was downregulated while MALAT1 and miR-142-3p were upregulated significantly in IBD group. GARP and MALAT1 expressions positively correlated in controls. MALAT1 and miR-142-3p expressions positively correlated in IBD group. MALAT1 was downregulated in aged HC but upregulated with smoking history across groups. No correlations occurred between gene expression and gender, diet, infections, or disease activity scores. CONCLUSIONS: Dysregulation of GARP, MALAT1, and miR-142-3p likely contributes to inflammation in IBD by reducing TGF-ß. MALAT1 is linked to smoking and age-related changes. These genes have potential as diagnostic markers or therapeutic targets for personalized IBD treatment.

STAT3 is a genetic modifier of TGF-beta induced EMT in KRAS mutant pancreatic cancer.

Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here, we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of the STAT3-responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.

Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells.

The primary site of metastasis for epithelial ovarian cancer (EOC) is the peritoneum, and it occurs through a multistep process that begins with adhesive contacts between cancer cells and mesothelial cells. Despite evidence that Notch signaling has a role in ovarian cancer, it is unclear how exactly it contributes to ovarian cancer omental metastasis, as well as the cellular dynamics and intrinsic pathways that drive this tropism. Here we show that tumor cells produced the Notch ligand Jagged2 is a clinically and functionally critical mediator of ovarian cancer omental metastasis by activating the Notch signaling in single-layered omental mesothelial cells. In turn, Jagged2 promotes tumor growth and therapeutic resistance by stimulating IL-6 release from mesothelial cells. Additionally, Jagged2 is a potent downstream mediator of the omental metastasis cytokine TGF-ß that is released during omental destruction. Importantly, therapeutic inhibition of Jagged2-mediated omental metastasis was significantly improved by directly disrupting the Notch pathway in omental mesothelial cells. These findings highlight the key role of Jagged2 to the functional interplay between the TGF-ß and the Notch signaling pathways during the metastatic process of ovarian cancer cells to the omentum and identify the Notch signaling molecule as a precision therapeutic target for ovarian cancer metastasis.

Investigation on the relationship between serum periostin, MMP-7, TGF-ß, and IL-18 levels and the clinical course and prognosis of COVID-19.

OBJECTIVE: Cytokines are involved in the inflammatory/anti-inflammatory balance and have been shown to play an important role in the course of COVID-19. This study aimed to evaluate the relationship of periostin, transforming growth factor-beta (TGF-ß), interleukin-18 (IL-18), and matrix metalloproteinase 7 (MMP-7) levels with clinical course and mortality in patients with early COVID-19 pneumonia. PATIENTS AND METHODS: A total of 150 hospitalized patients were diagnosed with COVID-19 between June and October 2021, and a control group of 30 healthy individuals were included in our study. The COVID-19 patients were divided into those who developed macrophage activation syndrome (MAS) in Group 1 and those who did not in Group 2. Serum periostin, MMP-7, TGF-ß, and IL-18 levels were measured from blood samples obtained at admission using enzyme-linked immunosorbent assay (ELISA). RESULTS: Periostin, MMP-7, and IL-18 levels were significantly higher in COVID-19 patients compared to the control group (p<0.001 for all). Periostin and MMP-7 levels were also significantly higher in Group 1 than in Group 2 (p<0.001 for both). Periostin, MMP-7, IL-18, and TGF-ß levels were significantly higher in non-surviving patients compared to survivors (p=0.04, p<0.001, p<0.001, and p<0.001, respectively). In the receiver operating characteristic (ROC) curve analysis, MMP-7 was found to have high sensitivity (90%) at a predictive value of 2.66 ng/mL. CONCLUSIONS: It is still not possible to predict which patients with early COVID-19 pneumonia will go on to develop MAS despite receiving standard treatment. The results of our study suggest that elevation of periostin and MMP-7 levels in the early period may predict the development of macrophage activation syndrome.

F127-SE-tLAP thermosensitive hydrogel alleviates bleomycin-induced skin fibrosis via TGF-ß/Smad pathway.

BACKGROUND: Skin fibrosis affects the normal function of the skin. TGF-ß1 is a key cytokine that affects organ fibrosis. The latency-associated peptide (LAP) is essential for TGF-ß1 activation. We previously constructed and prepared truncated LAP (tLAP), and confirmed that tLAP inhibited liver fibrosis by affecting TGF-ß1. SPACE peptide has both transdermal and transmembrane functions. SPACE promotes the delivery of macromolecules through the stratum corneum into the dermis. This study aimed to alleviate skin fibrosis through the delivery of tLAP by SPACE. METHODS: The SPACE-tLAP (SE-tLAP) recombinant plasmid was constructed. SE-tLAP was purified by nickel affinity chromatography. The effects of SE-tLAP on the proliferation, migration, and expression of fibrosis-related and inflammatory factors were evaluated in TGF-ß1-induced NIH-3T3 cells. F127-SE-tLAP hydrogel was constructed by using F127 as a carrier to load SE-tLAP polypeptide. The degradation, drug release, and biocompatibility of F127-SE-tLAP were evaluated. Bleomycin was used to induce skin fibrosis in mice. HE, Masson, and immunohistochemistry were used to observe the skin histological characteristics. RESULTS: SE-tLAP inhibited the proliferation, migration, and expression of fibrosis-related and inflammatory factors in NIH-3T3 cells. F127-SE-tLAP significantly reduced ECM production, collagen deposition, and fibrotic pathological changes, thereby alleviating skin fibrosis. CONCLUSION: F127-SE-tLAP could increase the transdermal delivery of LAP, reduce the production and deposition of ECM, inhibit the formation of dermal collagen fibers, and alleviate the progression of skin fibrosis. It may provide a new idea for the therapy of skin fibrosis.

The effect of saraglitazar on TGF-ß-induced smad3 phosphorylation and expression of genes related to liver fibrosis in LX2 cell line.

BACKGROUND AND PURPOSE: Liver fibrosis is a reversible liver injury that occurs as a result of many chronic inflammatory diseases and can lead to cirrhosis, which is irreversible and fatal. So, we studied the anti-fibrotic effects of saroglitazar on LX-2 cell lines, as a dual PPARα/γ agonist. METHODS: Cells, after 80% confluence, were treated with TGF-ß (2 ng/mL) for 24 h. Then cells were treated with saroglitazar at different doses (2.5, 5, 10 µM) for 24 h. After same incubation, the cells of control group, TGF-ß group, and TGF-ß + saroglitazar group were harvested for RNA and protein extraction to determine the effects of saroglitazar. RT-PCR and western blot methods were used to express genes related to fibrosis. RESULTS: Our results show that the relative expression of α-SMA, collagen1α, N-cadherin, NOX (1, 2, and 4), and phosphorylated Smad3 protein was significantly higher in TGF-ß-treated cells compared with the normal group, and E-cadherin expression was decreased in TGF-ß-treated cells. After TGF-ß-treated cells were exposed to saroglitazar, the expression of these genes was significantly reversed (P < 0.05). CONCLUSIONS: Our results clearly show the short-term inhibitory role of saroglitazar in the expression of fibrotic factors using the TGF-ß/Smad signaling pathway. These results suggest that saroglitazar can be considered as a suitable therapeutic strategy for fibrotic patients. Although more studies are needed.

Hydrostatic pressure mediates epithelial-mesenchymal transition of cholangiocytes through RhoA/ROCK and TGF-ß/smad pathways.

Biomechanical cue within the tissue microenvironment is known to play a critical role in regulating cell behaviors and maintaining tissue homeostasis. As hydrostatic pressure often increases in biliary system under pathological states, we investigated the effect of the moderate elevation of the hydrostatic pressure on biliary epithelial cells, especially on the epithelial-mesenchymal transition (EMT). Human intrahepatic biliary epithelial cells were loaded to hydrostatic pressure using a commercial device. We found that loading the cells to 50 mmHg hydrostatic pressure induced obvious morphological changes and significantly upregulated vimentin, ZEB1, and pSmad2/3, fibronectin, and collagen 1α. All changes induced by hydrostatic pressure loading were effectively mitigated by either ROCK inhibitor (Y-27632) or ALK5 inhibitor (SB-431542). Our in vitro experimental data suggests that hydrostatic pressure loading induces EMT of cholangiocytes through RhoA/ROCK and TGF-ß/Smad pathways. Elevated hydrostatic pressure in biliary duct system under pathological states may promote the biliary epithelial cells shifting to profibrotic and mesenchymal characteristics.

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept.

BACKGROUND: In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-ß is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-ß can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-ß receptors during MSC in vitro chondrogenesis. METHODS: Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-ß and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. RESULTS: All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. CONCLUSIONS: Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-ß that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-ß-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

Combination of Fushengong decoction with Western medicine on patients with chronic renal failure: An observational study.

Chronic renal failure (CRF) causes a reduction in glomerular filtration rate and damage to renal parenchyma. Fushengong decoction (FSGD) showed improvement in renal function in CRF rats. This study aims to analyze the differentially expressed proteins in CRF patients treated with Western medicine alone or in combination with FSGD. Sixty patients with CRF recruited from Yongchuan Traditional Chinese Medicine Hospital affiliated to Chongqing Medical University were randomly assigned into control (treated with Western medicine alone) and observation groups (received additional FSGD treatment thrice daily for 8 weeks). The clinical efficacy and changes in serum Bun, serum creatinine, Cystatin C, and transforming growth factor beta 1 (TGF-ß1) before and after treatment were observed. We employed isotope relative labeling absolute quantification labeling and liquid chromatography-mass spectrometry to identify differentially expressed proteins and carried out bioinformatics Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Patients in the observation group showed greater clinical improvement and lower levels of serum Bun, serum creatinine, Cyc-c, and TGF-ß1 than the control group. We identified 32 differentially up-regulated and 52 down-regulated proteins in the observation group. These proteins are involved in the blood coagulation system, protein serine/threonine kinase activity, and TGF-ß, which are closely related to the pathogenesis of CRF. Protein-protein-interaction network analysis indicated that candidate proteins fibronectin 1, fibrinogen alpha chain, vitronectin, and Serpin Family C Member 1 were in the key nodes. This study provided an experimental basis suggesting that FSGD combined with Western medicine could significantly improve renal function and renal fibrosis of CRF patients, which may be through the regulation of fibronectin 1, fibrinogen alpha chain, vitronectin, Serpin Family C Member 1, TGF-ß, and the complement coagulation pathway (see Graphical abstract S1, Supplemental Digital Content, http://links.lww.com/MD/L947).

Isolation and Single-Cell Transcriptomic Analysis of Murine Regulatory B Cells.

Regulatory B (Breg) cells have been demonstrated to play an important role in the inhibition of a wide range of immunological responses, and they are absent or malfunction in autoimmune diseases like lupus. Breg cells can control immunological responses and keep the immune system in a balanced state by releasing immunosuppressive cytokines such as transforming growth factor-beta (TGF-ß) and interleukin-10 (IL-10), which in turn promote regulatory T (Treg) cells and reduce effector T cell responses. Breg cells have also been linked to the modulation of cancer immunity. Due to their immunosuppressive role, in the context of cancer, Breg cells aid in tumor immune evasion and promote tumor progression. Nonetheless, it has been established that Breg cells are involved in both cancer immunity and autoimmunity, and their characterizations beyond surface markers, for example, on the transcriptomic level, are essential for our understanding of Breg biology in health and disease. In this chapter, using lupus-prone MRL/lpr mice, we describe a Breg cell isolation protocol for the purpose of single-cell RNA sequencing analysis.

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

Specific isoforms of the ubiquitin ligase gene WWP2 are targets of osteoarthritis genetic risk via a differentially methylated DNA sequence.

BACKGROUND: Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFß signaling. METHODS: Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments. RESULTS: rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140. CONCLUSIONS: As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFß signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.

Inhibition of MERTK reduces organ fibrosis in mouse models of fibrotic disease.

Transforming growth factor-ß (TGFß) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFß signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFß-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFß expression and drove TGFß signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFß signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.

Targeted inhibition of transforming growth factor-ß type I receptor by AZ12601011 improves paraquat poisoning-induced multiple organ fibrosis.

Paraquat (PQ) causes fatal poisoning that leads to systemic multiple organ fibrosis, and transforming growth factor (TGF)-ß1 plays a critical role in this process. In this study, we aimed to investigate the effects of AZ12601011 (a small molecular inhibitor of TGFßRI) on PQ-induced multiple organ fibrosis. We established a mouse model of PQ in vivo and used PQ-treated lung epithelial cell (A549) and renal tubular epithelial cells (TECs) in vitro. Haematoxylin-eosin and Masson staining revealed that AZ12601011 ameliorated pulmonary, hepatic, and renal fibrosis, consistent with the decrease in the levels of fibrotic indicators, alpha-smooth muscle actin (α-SMA) and collagen-1, in the lungs and kidneys of PQ-treated mice. In vitro data showed that AZ12601011 suppressed the induction of α-SMA and collagen-1 in PQ-treated A549 cells and TECs. In addition, AZ12601011 inhibited the release of inflammatory factors, interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α. Mechanistically, TGF-ß and TGFßRI levels were significantly upregulated in the lungs and kidneys of PQ-treated mice. Cellular thermal shift assay and western blotting revealed that AZ12601011 directly bound with TGFßRI and blocked the activation of Smad3 downstream. In conclusion, our findings revealed that AZ12601011 attenuated PQ-induced multiple organ fibrosis by blocking the TGF-ß/Smad3 signalling pathway, suggesting its potential for PQ poisoning treatment.

HWJMSC-EVs promote cartilage regeneration and repair via the ITGB1/TGF-ß/Smad2/3 axis mediated by microfractures.

The current first-line treatment for repairing cartilage defects in clinical practice is the creation of microfractures (MF) to stimulate the release of mesenchymal stem cells (MSCs); however, this method has many limitations. Recent studies have found that MSC-derived extracellular vesicles (MSC-EVs) play an important role in tissue regeneration. This study aimed to verify whether MSC-EVs promote cartilage damage repair mediated by MFs and to explore the repair mechanisms. In vitro experiments showed that human umbilical cord Wharton's jelly MSC-EVs (hWJMSC-EVs) promoted the vitality of chondrocytes and the proliferation and differentiation ability of bone marrow-derived MSCs. This was mainly because hWJMSC-EVs carry integrin beta-1 (ITGB1), and cartilage and bone marrow-derived MSCs overexpress ITGB1 after absorbing EVs, thereby activating the transforming growth factor-ß/Smad2/3 axis. In a rabbit knee joint model of osteochondral defect repair, the injection of different concentrations of hWJMSC-EVs into the joint cavity showed that a concentration of 50 µg/ml significantly improved the formation of transparent cartilage after MF surgery. Extraction of regenerated cartilage revealed that the changes in ITGB1, transforming growth factor-ß, and Smad2/3 were directly proportional to the repair of regenerated cartilage. In summary, this study showed that hWJMSC-EVs promoted cartilage repair after MF surgery.

Protein Disulfide Isomerase 4 Is an Essential Regulator of Endothelial Function and Survival.

Endothelial autophagy plays an important role in the regulation of endothelial function. The inhibition of endothelial autophagy is associated with the reduced expression of protein disulfide isomerase 4 (PDIA-4); however, its role in endothelial cells is not known. Here, we report that endothelial cell-specific loss of PDIA-4 leads to impaired autophagic flux accompanied by loss of endothelial function and apoptosis. Endothelial cell-specific loss of PDIA-4 also induced marked changes in endothelial cell architecture, accompanied by the loss of endothelial markers and the gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition (EndMT). The loss of PDIA-4 activated TGFß-signaling, and inhibition of TGFß-signaling suppressed EndMT in PDIA-4-silenced endothelial cells in vitro. Our findings help elucidate the role of PDIA-4 in endothelial autophagy and endothelial function and provide a potential target to modulate endothelial function and/or limit autophagy and EndMT in (patho-)physiological conditions.

FBXL8 inhibits post-myocardial infarction cardiac fibrosis by targeting Snail1 for ubiquitin-proteasome degradation.

Abnormal cardiac fibrosis is the main pathological change of post-myocardial infarction (MI) heart failure. Although the E3 ubiquitin ligase FBXL8 is a key regulator in the cell cycle, cell proliferation, and inflammation, its role in post-MI ventricular fibrosis and heart failure remains unknown. FBXL8 was primarily expressed in cardiac fibroblasts (CFs) and remarkably decreased in CFs treated by TGFß and heart subjected to MI. The echocardiography and histology data suggested that adeno-associated viruses (AAV9)-mediated FBXL8 overexpression had improved cardiac function and ameliorated post-MI cardiac fibrosis. In vitro, FBXL8 overexpression prevented TGFß-induced proliferation, migration, contraction, and collagen secretion in CFs, while knockdown of FBXL8 demonstrated opposite effects. Mechanistically, FBXL8 interacted with Snail1 to promote Snail1 degradation through the ubiquitin-proteasome system and decreased the activation of RhoA. Moreover, the FBXL8ΔC3 binding domain was indispensable for Snail1 interaction and degradation. Ectopic Snail1 expression partly abolished the effects mediated by FBXL8 overexpression in CFs treated by TGFß. These results characterized the role of FBXL8 in regulating the ubiquitin-mediated degradation of Snail1 and revealed the underlying molecular mechanism of how MI up-regulated the myofibroblasts differentiation-inducer Snail1 and suggested that FBXL8 may be a potential curative target for improving post-MI cardiac function.

Modular organization of functional brain networks in patients with degenerative cervical myelopathy.

Previous studies have indicated that brain functional plasticity and reorganization in patients with degenerative cervical myelopathy (DCM). However, the effects of cervical cord compression on the functional integration and separation between and/or within modules remain unclear. This study aimed to address these questions using graph theory. Functional MRI was conducted on 46 DCM patients and 35 healthy controls (HCs). The intra- and inter-modular connectivity properties of the whole-brain functional network and nodal topological properties were then calculated using theoretical graph analysis. The difference in categorical variables between groups was compared using a chi-squared test, while that between continuous variables was evaluated using a two-sample t-test. Correlation analysis was conducted between modular connectivity properties and clinical parameters. Modules interaction analyses showed that the DCM group had significantly greater inter-module connections than the HCs group (DMN-FPN: t = 2.38, p = 0.02); inversely, the DCM group had significantly lower intra-module connections than the HCs group (SMN: t = - 2.13, p = 0.036). Compared to HCs, DCM patients exhibited higher nodal topological properties in the default-mode network and frontal-parietal network. In contrast, DCM patients exhibited lower nodal topological properties in the sensorimotor network. The Japanese Orthopedic Association (JOA) score was positively correlated with inter-module connections (r = 0.330, FDR p = 0.029) but not correlated with intra-module connections. This study reported alterations in modular connections and nodal centralities in DCM patients. Decreased nodal topological properties and intra-modular connection in the sensory-motor regions may indicate sensory-motor dysfunction. Additionally, increased nodal topological properties and inter-modular connection in the default mode network and frontal-parietal network may serve as a compensatory mechanism for sensory-motor dysfunction in DCM patients. This could provide an implicative neural basis to better understand alterations in brain networks and the patterns of changes in brain plasticity in DCM patients.

First-in-human study of GFH018, a small molecule inhibitor of transforming growth factor-ß receptor I inhibitor, in patients with advanced solid tumors.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a cytokine with multiple functions, including cell growth regulation, extracellular matrix production, angiogenesis homeostasis adjustment and et al. TGF-ß pathway activation promotes tumor metastasis/progression and mediates epithelial-mesenchymal transmission suppressing immunosurveillance in advanced tumors. GFH018, a small molecule inhibitor blocking TGF-ß signal transduction, inhibits the progression and/or metastasis of advanced cancers. This first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and efficacy of GFH018 monotherapy in patients with advanced solid tumors. METHODS: This phase I, open-label, multicenter study used a modified 3+3 dose escalation and expansion design. Adult patients with advanced solid tumors failing the standard of care were enrolled. Starting at 5 mg, eight dose levels up to 85 mg were evaluated. Patients received GFH018 BID (14d-on/14d-off) starting on the 4th day after a single dose on cycle 1, day 1. Subsequent cycles were defined as 28 days. The study also explored the safety of 85 mg BID 7d-on/7d-off. Adverse events were graded using NCI criteria for adverse events (NCI-CTCAE v5.0). PK was analyzed using a noncompartmental method. Efficacy was evaluated using RECIST 1.1. Blood samples were collected for biomarker analysis. RESULTS: Fifty patients were enrolled and received at least one dose of GFH018. No dose-limiting toxicity occurred, and the maximum tolerated dose was not reached. Forty-three patients (86.0%) had at least one treatment-related adverse event (TRAE), and three patients (6.0%) had ≥ G3 TRAEs. The most common TRAEs (any grade/grade ≥3) were AST increased (18%/0%), proteinuria (14%/2%), anemia (14%/2%), and ALT increased (12%/0%). No significant cardiotoxicity or bleeding was observed. GFH018 PK was linear and dose-independent, with a mean half-life of 2.25-8.60 h from 5 - 85 mg. Nine patients (18.0%) achieved stable disease, and one patient with thymic carcinoma achieved tumor shrinkage, with the maximum target lesion decreased by 18.4%. Serum TGF-ß1 levels were not associated with clinical responses. The comprehensive recommended dose for Phase II was defined as 85 mg BID 14d-on/14d-off. CONCLUSIONS: GFH018 monotherapy presented a favorable safety profile without cardiac toxicity or bleeding. Modest efficacy warrants further studies, including combination strategies. TRIAL REGISTRATION: ClinicalTrial. gov ( https://www. CLINICALTRIALS: gov/ ), NCT05051241. Registered on 2021-09-02.