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Alterations in the Wnt signaling pathway including those impacting hepatic stellate cells (HSCs) have been implicated in liver fibrosis. In the current study, we first examined the expression of Wnt genes in human HSC (HHSCs) after treatment with a profibrogenic factor TGF-β1. Next, we generated HSC-specific Wntless (Wls) knockout (KO) using the Lrat-cre and Wls-floxed mice. KO and littermate controls (CON) were characterized for any basal phenotype and subjected to two liver fibrosis protocols. In vitro, TGF-β1 induced expression of Wnt2, 5a and 9a while decreasing Wnt2b, 3a, 4, and 11 in HHSC. In vivo, KO and CON mice were born at normal Mendelian ratio and lacked any overt phenotype. Loss of Wnt secretion from HSCs had no effect on liver weight and did not impact β-catenin activation in the pericentral hepatocytes. After 7 days of bile duct ligation (BDL), KO and CON showed comparable levels of serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total and direct bilirubin. Comparable histology, Sirius red staining, and immunohistochemistry for α-SMA, desmin, Ki-67, F4/80, and CD45 indicated similar proliferation, inflammation, and portal fibrosis in both groups. Biweekly administration of carbon tetrachloride for 4 or 8 weeks also led to comparable serum biochemistry, inflammation, and fibrosis in KO and CON. Specific Wnt genes were altered in HHSCs in response to TGF-β1; however, eliminating Wnt secretion from HSC did not impact basal β-catenin activation in normal liver nor did it alter the injury-repair response during development of liver fibrosis.

Background Retinitis pigmentosa (RP) is a hereditary retinal disease which leads to visual impairment. The onset and progression of RP has physiological consequences that affects the ocular environment. Some of the key non-genetic factors which hasten the retinal degeneration in RP include oxidative stress, hypoxia and ocular inflammation. In this study, we investigated the status of the ocular immune privilege during retinal degeneration and the effect of ocular immune changes on the peripheral immune system in RP. We assessed the peripheral blood mononuclear cell stimulation by retinal antigens and their immune response status in RP patients. Subsequently, we examined alterations in ocular immune privilege machineries which may contribute to ocular inflammation and disease progression in rd1 mouse model. Results In RP patients, we observed a suppressed anti-inflammatory response to self-retinal antigens, thereby indicating a deviated response to self-antigens. The ocular milieu in rd1 mouse model indicated a significant decrease in immune suppressive ligands and cytokine TGF-B1, and higher pro-inflammatory ocular protein levels. Further, blood–retinal-barrier breakdown due to decrease in the expression of tight junction proteins was observed. The retinal breach potentiated pro-inflammatory peripheral immune activation against retinal antigens and caused infiltration of the peripheral immune cells into the ocular tissue. Conclusions Our studies with RP patients and rd1 mouse model suggest that immunological consequences in RP is a contributing factor in the progression of retinal degeneration. The ocular inflammation in the RP alters the ocular immune privilege mechanisms and peripheral immune response. These aberrations in turn create an auto-reactive immune environment and accelerate retinal degeneration.

ObjectiveReciprocal cellular crosstalk within the tumour microenvironment (TME) actively participates in tumour progression. The anterior gradient-2 (AGR2) can be secreted to extracellular compartments and contribute to colorectal cancer (CRC) metastasis. We investigated the cellular source for secreted AGR2 in the TME and underlying mechanisms mediating secreted AGR2’s effects.DesignTissue microarray, tumour tissues, blood samples and tumour-associated neutrophils (TANs) from patients with CRC were isolated for phenotypical and functional analyses. The role of TAN-secreted AGR2 was determined in neutrophil-specific Agr2 knockout (Agr2f/f;Mrp-Cre) mice. The biological roles and mechanisms of secreted AGR2 in CRC metastasis were determined in vitro and in vivo.ResultsTANs were a predominant cell type for secreting AGR2 in the TME of CRC. TANs-secreted AGR2 promoted CRC cells’ migration. Neutrophils-specific ablation of Agr2 in mice ameliorated CRC liver metastases. The heavy chain of CD98 (CD98hc) served as the functional receptor for secreted AGR2. Mechanistically, secreted AGR2 increased xCT activity in a CD98hc-dependent manner, subsequently activating Ras homologue family member A/Rho-associated protein kinase 2 cascade. CRC cells actively recruited TANs through the C-X-C motif chemokine 2. Moreover, CRC-derived transforming growth factor beta 1 (TGF-β1) educated peripheral blood neutrophils to become AGR2+ TANs that secrete AGR2. Abundant infiltration of AGR2+ TANs and high expression of TGF-β1 and CD98hc–xCT were correlated with poor prognosis of patients with CRC.ConclusionsOur study unveils a novel crosstalk between TANs and CRC cells involving the secreted AGR2–CD98hc–xCT axis that promotes metastasis and impacts the outcomes of patients with CRC.

The discovery of regulated cell death processes has enabled advances in cancer treatment. In the past decade, ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation, has been implicated in the development and therapeutic responses of various types of tumours. Experimental reagents (such as erastin and RSL3), approved drugs (for example, sorafenib, sulfasalazine, statins and artemisinin), ionizing radiation and cytokines (such as IFNγ and TGFβ1) can induce ferroptosis and suppress tumour growth. However, ferroptotic damage can trigger inflammation-associated immunosuppression in the tumour microenvironment, thus favouring tumour growth. The extent to which ferroptosis affects tumour biology is unclear, although several studies have found important correlations between mutations in cancer-relevant genes (for example, RAS and TP53), in genes encoding proteins involved in stress response pathways (such as NFE2L2 signalling, autophagy and hypoxia) and the epithelial-to-mesenchymal transition, and responses to treatments that activate ferroptosis. Herein, we present the key molecular mechanisms of ferroptosis, describe the crosstalk between ferroptosis and tumour-associated signalling pathways, and discuss the potential applications of ferroptosis in the context of systemic therapy, radiotherapy and immunotherapy.Ferroptosis is an iron-dependent form of regulated cell death driven by excessive lipid peroxidation. Pharmacological agents, ionizing radiation and cytokines can induce ferroptosis and thus suppress tumour growth, but ferroptosis can also trigger inflammation-associated immunosuppression. The authors describe the key molecular mechanisms of ferroptosis, including crosstalk with tumour-associated signalling pathways, and discuss potential therapeutic applications of ferroptosis.

BackgroundTranscription of transforming growth factor beta-1 (TGF-β1) is regulated by a polymorphic promoter region containing African-specific single nucleotide polymorphisms (SNPs). Some of these SNPs have higher frequencies among Southern Africans compared to other African populations and their functionality has only been partially studied. Due to the high prevalence of HIV-associated nephropathy (HIVAN) in Africans we hypothesized that functional African TGFB1-promoter SNPs may contribute to HIVAN pathogenesis.MethodsThe functionality of the TGFB1 -1347 C>T variant and African-specific variants (-1287 G>A, -1154 C>T, -387 C>T and -14 G>A) were examined by measuring reporter gene expression in kidney and fibroblast cell lines co-transfected with TGFB1-promoter constructs and an HIV-Tat expression vector. TGF-β1 immunohistochemical staining was performed on kidney biopsies with HIVAN (n = 18) and compared to control biopsies without HIVAN or tubulointerstitial disease (n = 12) using semi-quantitative and digital image analysis. HIVAN cases were genotyped for TGFB1 -1347 and -387 SNP variants.ResultsTGFB1-promoter haplotypes containing the African -387 T-allele resulted in ~ five-fold repression of TGFB1-promoter activity compared to -387 C haplotypes (p ≤ 0.024). HIV-Tat upregulated TGFB1-promoter activity for haplotypes containing -1347 T and -387 T in transfected renal cells (≈ 1.6-fold; p ≤ 0.030) and fibroblasts (≈ 1.3-fold; p ≤ 0.016). The renal interstitium from HIVAN biopsies, compared to HIV-positive and -negative controls, differed in the semi-quantitative TGF-β1 staining and digital optical density analyses. The TGFB1 -1347 and -387 genotypes in HIVAN cases were similar to population controls.ConclusionAfrican-specific haplotypes lower TGFB1-promoter activity and expression levels and HIV-Tat upregulates TGFB1 promoter activity irrespective of the haplotype.

Osteoarthritis affects millions of people worldwide but current treatments using analgesics or anti-inflammatory drugs only alleviate symptoms of this disease. Here, we present an injectable, biodegradable piezoelectric hydrogel, made of short electrospun poly-L-lactic acid nanofibers embedded inside a collagen matrix, which can be injected into the joints and self-produce localized electrical cues under ultrasound activation to drive cartilage healing. In vitro, data shows that the piezoelectric hydrogel with ultrasound can enhance cell migration and induce stem cells to secrete TGF-β1, which promotes chondrogenesis. In vivo, the rabbits with osteochondral critical-size defects receiving the ultrasound-activated piezoelectric hydrogel show increased subchondral bone formation, improved hyaline-cartilage structure, and good mechanical properties, close to healthy native cartilage. This piezoelectric hydrogel is not only useful for cartilage healing but also potentially applicable to other tissue regeneration, offering a significant impact on the field of regenerative tissue engineering. The use of biomaterial scaffolds-based cartilage grafts could potentially innovate the Osteoarthritis (OA) treatment, but has been limited by toxicity concerns and invasive surgical procedures. Here, the authors report an injectable and biodegradable piezoelectric hydrogel with ultrasound activation to offer a minimally invasive approach for OA treatment.

Despite orientationally variant tears of the meniscus, suture repair is the current clinical gold treatment. However, inaccessible tears in company with re-tears susceptibility remain unresolved. To extend meniscal repair tools from the perspective of adhesion and regeneration, we design a dual functional biologic-released bioadhesive (S-PIL10) comprised of methacrylated silk fibroin crosslinked with phenylboronic acid-ionic liquid loading with growth factor TGF-β1, which integrates chemo-mechanical restoration with inner meniscal regeneration. Supramolecular interactions of β-sheets and hydrogen bonds richened by phenylboronic acid-ionic liquid (PIL) result in enhanced wet adhesion, swelling resistance, and anti-fatigue capabilities, compared to neat silk fibroin gel. Besides, elimination of reactive oxygen species (ROS) by S-PIL10 further fortifies localized meniscus tear repair by affecting inflammatory microenvironment with dynamic borate ester bonds, and S-PIL10 continuously releases TGF-β1 for cell recruitment and bridging of defect edge. In vivo rabbit models functionally evidence the seamless and dense reconstruction of torn meniscus, verifying that the concept of meniscus adhesive is feasible and providing a promising revolutionary strategy for preclinical research to repair meniscus tears. Suture repair is the current clinical treatment for meniscus tears, but inaccessible tears in company with re-tears susceptibility remain unresolved. Here the authors address these issues by developing a meniscus adhesive-based strategy for the seamless and dense reconstruction of torn meniscus.

Abstract Pirfenidone (PFD) is a pharmacological compound with therapeutic efficacy in idiopathic pulmonary fibrosis. It has been chiefly characterized as an antifibrotic agent, although it was initially developed as an antiinflammatory compound because of its ability to diminish the accumulation of inflammatory cells and cytokines. Despite recent studies that have elucidated key mechanisms, the precise molecular activities of PFD remain incompletely understood. PFD modulates fibrogenic growth factors, thereby attenuating fibroblast proliferation, myofibroblast differentiation, collagen and fibronectin synthesis, and deposition of extracellular matrix. This effect is mediated by suppression of TGF-β1 (transforming growth factor-β1) and other growth factors. Here, we appraise the impact of PFD on TGF-β1 production and its downstream pathways. Accumulating evidence indicates that PFD also downregulates inflammatory pathways and therefore has considerable potential as a viable and innovative antiinflammatory compound. We examine the effects of PFD on inflammatory cells and the production of pro- and antiinflammatory cytokines in the lung. In this context, recent evidence that PFD can target inflammasome pathways and ensuing lung inflammation is highlighted. Finally, the antioxidant properties of PFD, such as its ability to inhibit redox reactions and regulate oxidative stress–related genes and enzymes, are detailed. In summary, this narrative review examines molecular mechanisms underpinning PFD and its recognized benefits in lung fibrosis. We highlight preclinical data that demonstrate the potential of PFD as a nonsteroidal antiinflammatory agent and outline areas for future research.

The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-β, BMP,Wnt–β-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-β1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-β–induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.