Smad3 Inhibitor Research Articles

Endothelial-to-Mesenchymal Transition Contributes to Accelerated Atherosclerosis in Hutchinson-Gilford Progeria Syndrome.

Atherosclerosis is the main medical problem in Hutchinson-Gilford progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe-deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear. Apoe- or Ldlr-deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFβ (transforming growth factor β) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFβ1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root. Both ubiquitous and VSMC-specific progerin expression in Apoe-null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFβ1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype. Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFβ1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis.

C-reactive protein promotes diabetic kidney disease via Smad3 mediated NLRP3 inflammasome activation

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney diseases resulting enormous social-economic burden. Accumulated evidence has indicated that C-reactive protein (CRP) exacerbates DKD by enhancing renal inflammation and fibrosis through TGF-β/Smad3 signaling. NLRP3 inflammasome is the key sensor contributing to renal inflammation. However, whether CRP enhances inflammation in DKD via NLRP3 inflammasome related pathway remains unknown. In this study, we demonstrate that CRP promotes DKD via Smad3-mediated NLRP3 inflammasome activation as mice overexpressing human CRP gene exhibits accelerated renal inflammation in diabetic kidneys, which is associated with the activation of Smad3 and NLRP3 inflammasome. In contrast, blockade of CPR signaling with a neutralizing anti-CD32 antibody attenuates CRP-induced activation of Smad3 and NLRP3 in vitro. Importantly, genetic deletion or pharmacological inhibition of Smad3 also mitigates CRP-induced activation of NLRP3 in diabetic kidneys or in high glucose treated cells. Mechanistically, we reveal that Smad3 binds to the NLRP3 gene promoter which is enhanced by CRP. Taken together, we conclude that CRP induces renal inflammation in DKD via Smad3-NLRP3 inflammasome-dependent mechanism. Thus, targeting CRP or Smad3-NLRP3 pathways may be a new therapeutic potential for DKD.

GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging.

Age-related susceptibility to sepsis and endotoxemia is poorly defined, although hyperactivation of the immune system and the expansion of the visceral adipose as an immunological reservoir are underlying features. Macrophages from older organisms exhibit substantial changes, including chronic NLRP3 inflammasome activation, genomic remodeling and a dysfunctional, amplified inflammatory response upon new exposure to pathogen. However, the mechanisms by which old macrophages maintain their inflammatory phenotype during endotoxemia remains elusive. We previously identified Gdf3 , a TGFβ superfamily cytokine, as a top-regulated gene by age and the NLRP3 inflammasome in adipose tissue macrophages (ATMs). Here, we demonstrate that endotoxemia increases inflammatory (CD11c + ) ATMs in a Gdf3- dependent manner in old mice. Lifelong systemic or myeloid-specific deletion of Gdf3 leads to reduced endotoxemia- induced inflammation, with decreased CD11c + ATMs and inflammatory cytokines, and protection from hypothermia. Moreover, acute blockade of Gdf3 using JQ1, a BRD4 inhibitor, phenocopies old mice with lifelong Gdf3- deficiency. We show that GDF3 promotes the inflammatory phenotype in ATMs by phosphorylating SMAD2/3. Mechanistically, the differential chromatin landscape of ATMs from old mice with or without myeloid-driven Gdf3 indicates that GDF3- SMAD2/3 signaling axis shifts the chromatin accessibility of ATMs towards an inflammatory state during aging. Furthermore, pharmaceutical inhibition of SMAD3 with a specific inhibitor of SMAD3 (SIS3) mimics Gdf3 deletion. SIS3 reduces endotoxemia-mediated inflammation with fewer CD11c + ATMs and less severe hypothermia in old, but not young mice, as well as reduced mortality. In human adipose tissue, age positively correlates with GDF3 level, while inflammation correlates with pSMAD2/3 level. Overall, these results highlight the importance of GDF3-SMAD2/3 axis in driving inflammation in older organisms and identify this signaling axis as a promising therapeutic target for mitigating endotoxemia-related inflammation in the aged.

Tanshinone IIA Loaded Inhaled Polymer Nanoparticles Alleviate Established Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by chronic, progressive scarring of the lung parenchyma, leading to an irreversible decline in lung function. Apart from supportive care, there is currently no specific treatment available to reverse the disease. Based on the fact that tanshinone IIA (TAN) had an effect on protecting against TGF-β1-induced fibrosis through the inhibition of Smad and non-Smad signal pathways to avoid myofibroblasts activation, this study reported the development of the inhalable tanshinone IIA-loaded chitosan-oligosaccharides-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CPN@TAN) for enhancing the pulmonary delivery of tanshinone IIA to treat pulmonary fibrosis. The CPN@TAN with a size of 206.5 nm exhibited excellent in vitro aerosol delivery characteristics, featuring a mass median aerodynamic diameter (MMAD) of 3.967 ± 0.025 μm and a fine particle fraction (FPF) of 70.516 ± 0.929%. Moreover, the nanoparticles showed good stability during atomization and enhanced the mucosal penetration capabilities. The results of confocal spectroscopy confirmed the potential of the nanoparticles as carriers that facilitated the uptake of drugs by NIH3T3, A549, and MH-S cells. Additionally, the nanoparticles demonstrated good in vitro biocompatibility. In a mouse model of bleomycin-induced pulmonary fibrosis, noninvasive inhalation of aerosol CPN@TAN greatly suppressed collagen formation and facilitated re-epithelialization of the destroyed alveolar epithelium without causing systemic toxicity compared with intravenous administration. Consequently, our noninvasive inhalation drug delivery technology based on polymers may represent a promising paradigm and open the door to overcoming the difficulties associated with managing pulmonary fibrosis.

Activin A plays an essential role in migration and proliferation of hepatic stellate cells via Smad3 and calcium signaling

Activin A and hepatic stellate cells (HSCs) are involved in tissue repair and fibrosis in liver injury. This study investigated the impact of activin A on HSC activation and migration. A microfluidic D4-chip was used for examining the cell migration of mouse hepatic stellate cell line MHSteC. The analysis of differentially expressed genes revealed that activin βA (Inhba), activin receptor type 1A (Acvr1a) and type 2A (Acvr2a) mRNAs were more significantly expressed in human HSCs than in the hepatocytes. Moreover, activin A promoted MHSteC proliferation and induced MHSteC migration. Furthermore, the MHSteCs treated with activin A exhibited increased levels of migration-related proteins, N-cadherin, Vimentin, α-SMA, MMP2 and MMP9, but a decreased level of E-cadherin. Additionally, activin A treatment significantly increased the p-Smad3 levels and p-Smad3/Smad3 ratio in the MHSteCs, and the Smad3 inhibitor SIS3 attenuated activin A-induced MHSteC proliferation and migration. Simultaneously, activin A increased the calcium levels in the MHSteCs, and the migratory effects of activin A on MHSteCs were weakened by the intracellular calcium ion-chelating agent BAPTA-AM. These data indicate that activin A can promote MHSteC activation and migration through the canonical Smad3 signaling and calcium signaling.

Antifibrotic and Pro-regenerative Effects of SMAD3 siRNA and Collagen I mRNA-Loaded Lipid Nanoparticles in Human Tenocytes.

Tendinopathy involves the inflammation and degeneration of the tendon due to repetitive strain injury. Current treatments primarily target inflammation resolution, yet they do not aim at tissue regeneration. In this study, a microfluidics approach is harnessed to develop a platform of lipid nanoparticles (LNPs) loaded simultaneously with SMAD3 siRNA and collagen I mRNA, aiming to explore its potential dual antifibrotic and regenerative effects in human tenocytes. The developed LNPs displayed size homogeneity and colloidal stability and exhibited high cytocompatibility in human tenocytes. Moreover, LNPs allowed for efficient uptake and transfection efficiency of the RNAs. In the in vitro efficacy studies, the gene expression and production of SMAD3 and collagen I were tested by real-time quantitative chain polymerase reaction and immuno- and intracellular staining, revealing collagen I production enhancement, SMAD3 inhibition, and modulation of other tendon repair factors by the LNPs. Overall, the potential of this platform of RNA-loaded LNPs to be used as a dual therapeutic approach to prevent fibrosis and promote tissue remodeling in late stages of tendon diseases was confirmed.

MiR-27b-3p reduces muscle fibrosis during chronic skeletal muscle injury by targeting TGF-βR1/Smad pathway

BackgroundFibrosis is a significant pathological feature of chronic skeletal muscle injury, profoundly affecting muscle regeneration. Fibro-adipogenic progenitors (FAPs) have the ability to differentiate into myofibroblasts, acting as a primary source of extracellular matrix (ECM). the process by which FAPs differentiate into myofibroblasts during chronic skeletal muscle injury remains inadequately explored.Methodmouse model with sciatic nerve denervated was constructed and miRNA expression profiles between the mouse model and uninjured mouse were analyzed. qRT/PCR and immunofluorescence elucidated the effect of miR-27b-3p on fibrosis in vivo and in vitro. Dual-luciferase reporter identified the target gene of miR-27b-3p, and finally knocked down or overexpressed the target gene and phosphorylation inhibition of Smad verified the influence of downstream molecules on the abundance of miR-27b-3p and fibrogenic differentiation of FAPs.ResultFAPs derived from a mouse model with sciatic nerves denervated exhibited a progressively worsening fibrotic phenotype over time. Introducing agomiR-27b-3p effectively suppressed fibrosis both in vitro and in vivo. MiR-27b-3p targeted Transforming Growth Factor Beta Receptor 1 (TGF-βR1) and the abundance of miR-27b-3p was negatively regulated by TGF-βR1/Smad.ConclusionmiR-27b-3p targeting the TGF-βR1/Smad pathway is a novel mechanism for regulating fibrogenic differentiation of FAPs. Increasing abundance of miR-27b-3p, suppressing expression of TGF-βR1 and inhibiting phosphorylation of smad3 presented potential strategies for treating fibrosis in chronic skeletal muscle injury.

Therapeutic targeting of PLK1 in TERT promoter-mutant hepatocellular carcinoma.

Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA-seq, CRISPR-Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit-8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V-FITC staining and/or propidium iodide staining. PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild-type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild-type nucleotide. Comparing the HCC cells with wild-type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti-tumor effect in TERT mutant HCC cells.

Cyclic strain alters the transcriptional and migratory response of scleral fibroblasts to TGFβ

In glaucoma, scleral fibroblasts are exposed to IOP-associated mechanical strain and elevated TGFβ levels. These stimuli, in turn, lead to scleral remodeling. Here, we examine the scleral fibroblast migratory and transcriptional response to these stimuli to better understand mechanisms of glaucomatous scleral remodeling. Human peripapillary scleral (PPS) fibroblasts were cultured on parallel grooves, treated with TGFβ (2 ng/ml) in the presence of vehicle or TGFβ signaling inhibitors, and exposed to uniaxial strain (1 Hz, 5%, 12–24 h). Axis of cellular orientation was determined at baseline, immediately following strain, and 24 h after strain cessation with 0° being completely aligned with grooves and 90° being perpendicular. Fibroblasts migration in-line and across grooves was assessed using a scratch assay. Transcriptional profiling of TGFβ-treated fibroblasts with or without strain was performed by RT-qPCR and pERK, pSMAD2, and pSMAD3 levels were measured by immunoblot. Pre-strain alignment of TGFβ-treated cells with grooves (6.2 ± 1.5°) was reduced after strain (21.7 ± 5.3°, p < 0.0001) and restored 24 h after strain cessation (9.5 ± 2.6°). ERK, FAK, and ALK5 inhibition prevented this reduction; however, ROCK, YAP, or SMAD3 inhibition did not. TGFβ-induced myofibroblast markers were reduced by strain (αSMA, POSTN, ASPN, MLCK1). While TGFβ-induced phosphorylation of ERK and SMAD2 was unaffected by cyclic strain, SMAD3 phosphorylation was reduced (p = 0.0004). Wound healing across grooves was enhanced by ROCK and SMAD3 inhibition but not ERK or ALK5 inhibition. These results provide insight into the mechanisms by which mechanical strain alters the cellular response to TGFβ and the potential signaling pathways that underlie scleral remodeling.

Can Asiatic Acid from Centella asiatica Be a Potential Remedy in Cancer Therapy?-A Review.

Centella asiatica has been recognized for centuries in Eastern medicine for its pharmacological properties. Due to the increasing prevalence of oncological diseases worldwide, natural substances that could qualify as anticancer therapeutics are becoming increasingly important subjects of research. This review aims to find an innovative use for asiatic acid (AA) in the treatment or support of cancer therapy. It has been demonstrated that AA takes part in inhibiting phosphorylation, inducing cell death, and reducing tumor growth and metastasis by influencing important signaling pathways, such as PI3K, Akt, mTOR, p70S6K, and STAT3, in cancer cells. It is also worth mentioning the high importance of asiatic acid in reducing the expression of markers such as N-cadherin, β-catenin, claudin-1, and vimentin. Some studies have indicated the potential of asiatic acid to induce autophagy in cancer cells through changes in the levels of specific proteins such as LC3 and p62. It can also act as an anti-tumor immunotherapeutic agent, thanks to its inductive effect on Smad7 in combination with naringenin (an Smad3 inhibitor). It seems that asiatic acid may be a potential anticancer drug or form of adjunctive therapy. Further studies should take into account safety and toxicity issues, as well as limitations related to the pharmacokinetics of AA and its low oral bioavailability.

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4559.

SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

Knocking out USP7 attenuates cardiac fibrosis and endothelial-to-mesenchymal transition by destabilizing SMAD3 in mice with heart failure with preserved ejection fraction.

Background: Heart failure with preserved ejection fraction (HFpEF) is a predominant type of heart failure. Exploring new pathogenesis and identifying potential novel therapeutic targets for HFpEF is of paramount importance. Methods: HFpEF mouse model was established by the "Multiple-hit" strategy, in that 18- to 22-month-old female C57B6/J mice fed with a high-fat diet were further challenged with chronic infusion of Angiotensin II. RNA sequencing analysis showed that USP7 was significantly increased in the heart of HFpEF mice. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis, in conjunction with co-immunoprecipitation (Co-IP) techniques, identified expression of SMAD3, the key molecule of endothelial-to-mesenchymal transition (EndMT), was also significantly elevated. USP7 endothelium-specific knockout mice was generated to investigate the involvement of USP7 in HFpEF. The biological significance of the interaction between USP7 and SMAD3 was further explored. Results: USP7 promotes EndMT and cardiac fibrosis by binding to SMAD3 directly via its UBL (Ubiquitin-like) domain and cysteine at position 223 of USP7, leading SMAD3 deubiquitination to maintain the stability of SMAD3 by removing the K63 ubiquitin chain and preventing the degradation of SMAD3 by proteasomal process. USP7 also promotes SMAD3 phosphorylation and nuclear translocation, thereby aggravating EndMT and cardiac fibrosis. Endothelium-specific USP7 knockout led to improvement of HFpEF phenotypes and reduction of cardiac fibrosis. Overexpression of SMAD3 in endothelium-specific knockout HFpEF mice reversed the protective effects of USP7 knockout in this HFpEF mouse model. Conclusion: Our results indicated that USP7 is one of the key pathogenic molecules of HFpEF, and knocking out USP7 could attenuate HFpEF injury by promoting the degradation of SMAD3. USP7 and SMAD3 inhibition might be potential therapeutic options for HFpEF.

Atractylenolide II combined with Interferon-γ synergistically ameliorates colorectal cancer progression in vivo and in vitro by blocking the NF-kB p65/PD-L1 pathway.

Purpose: Atractylodes macrocephala Koidz is a widely used classical traditional Chinese herbal medicine, that has shown remarkable efficacy in cancers. Colorectal cancer (CRC) is the most common malignant tumor globally. Interferon (IFN)-γ, a prominent cytokine involved in anti-tumor immunity that has cytostatic, pro-apoptotic, and immune-stimulatory properties for the detection and removal of transformed cells. Atractylenolides-II (AT-II) belongs to the lactone compound that is derived from Atractylodes macrocephala Koidz with anti-cancer activity. However, whether AT-II combined with IFN-γ modulates CRC progression and the underlying mechanisms remain unclear. The present study aimed to elucidate the efficacy and pharmaceutical mechanism of action of AT-II combined with IFN-γ synergistically against CRC by regulating the NF-kB p65/PD-L1 signaling pathway. Methods: HT29 and HCT15 cells were treated with AT-II and IFN-γ alone or in combination and cell viability, migration, and invasion were then analyzed using Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Furthermore, the underlying mechanism was investigated through western blot assay. The role of AT-II combined with IFN-γ on tumor growth and lung metastases was estimated in vivo. Finally, the population of lymphocytes in tumor tissues of lung metastatic C57BL/6 mice and the plasma cytokine levels were confirmed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Results: AT-II or the combination IFN-γ significantly inhibited the growth and migration abilities of CRC cells in vitro and in vivo. The biological mechanisms behind the beneficial effects of AT-II combined with IFN-γ were also measured and inhibition of p38 MAPK, FAK, Wnt/β-catenin, Smad, and NF-kB p65/PD-L1 pathways was observed. Moreover, AT-II combined with IFN-γ significantly inhibited HCT15 xenograft tumor growth and lung metastases in C57BL/6 mice, which was accompanied by lymphocyte infiltration into the tumor tissues and inflammatory response inactivation. Conclusions: The results showed that the AT-II in combination with IFN-γ could be used as a potential strategy for tumor immunotherapy in CRC. More importantly, the mechanism by which AT-II suppressed CRC progressions was by inhibiting the NF-kB p65/PD-L1 signal pathway.

Treatment for type 2 diabetes and diabetic nephropathy by targeting Smad3 signaling.

TGF-β/Smad3 signaling plays a critical role in type 2 diabetes (T2D) and type 2 diabetic nephropathy (T2DN), but treatment by specifically targeting Smad3 remains unexplored. To develop a new Smad3-targeted therapy for T2D and T2DN, we treated db/db mice at the pre-diabetic or established diabetic stage with a pharmacological Smad3 inhibitor SIS3. The therapeutic effect and mechanisms of anti-Smad3 treatment on T2D and T2DN were investigated. We found that anti-Smad3 treatment on pre-diabetic db/db mice largely attenuated both T2D and T2DN by markedly reducing blood glucose levels, and inhibiting the elevated serum creatinine, microalbuminuria, and renal fibrosis and inflammation. Unexpectedly, although SIS3 treatment on the established diabetic db/db mice inhibited T2DN but did not significantly improve T2D. Mechanistically, we uncovered that inhibition of T2DN in SIS3-treated db/db mice was associated with effectively restoring the balance of TGF-β/Smad signaling by inhibiting Smad3 while increasing Smad7, thereby suppressing Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation via lncRNA Erbb4-IR and LRN9884-dependent mechanisms. We also revealed that inhibition of islet β cell injury by preventing the loss of islet Pax 6 could be the mechanism through which the pre-diabetic treatment, rather than the late SIS3 treatment on db/db mice significantly improved the T2D phenotype.

Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas.

Recent studies have demonstrated the impact of pro-inflammatory signaling and reactive microglia/macrophages on the formation of Müller glial-derived progenitor cells (MGPCs) in the retina. In chick retina, ablation of microglia/macrophages prevents the formation of MGPCs. Analyses of single-cell RNA-sequencing chick retinal libraries revealed that quiescent and activated microglia/macrophages have a significant impact upon the transcriptomic profile of Müller glia (MG). In damaged monocyte-depleted retinas, MG fail to upregulate genes related to different cell signaling pathways, including those related to Wnt, heparin-binding epidermal growth factor (HBEGF), fibroblast growth factor (FGF) and retinoic acid receptors. Inhibition of GSK3β, to simulate Wnt signaling, failed to rescue the deficit in MGPC formation, whereas application of HBEGF or FGF2 completely rescued the formation of MGPCs in monocyte-depleted retinas. Inhibition of Smad3 or activation of retinoic acid receptors partially rescued the formation of MGPCs in monocyte-depleted retinas. We conclude that signals produced by reactive microglia/macrophages in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming of MG into proliferating MGPCs.

IL-37 counteracts inflammatory injury in the temporomandibular joint via the intracellular pathway.

Background: The temporomandibular joint is often afflicted by osteoarthritis (TMJOA), causing pain and dysfunction, which is particularly prevalent in the elderly population. IL-37 is effective in avoiding excessive inflammatory damage to the organism. This article investigates the role and mechanism of intracellular IL-37 in TMJOA. Methods: Enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, Senescence-associated β-galactosidase staining, immunofluorescence, and lentivirus were performed to elucidate the underlying mechanism. Results: The results confirmed that IL-37 in synovial cells decreased with aging. Inflammatory stimulus elevated intracellular IL-37 in synoviocytes, while lentiviral knockdown of IL-37 resulted in more inflammatory factor production. Dynamic changes of IL-37 were observed in the nucleus and supernatant. In addition, Caspease-1 inhibitor hindered intracellular IL-37 maturation, and Smad3 inhibitor caused the loss of nuclear translocation of mature IL-37. Transfection of synovial cells with IL-37-expressing lentivirus resulted in relief not only of synovitis but also of the cartilage damage and inflammation caused by synovitis. Conclusion: This study provides new insights into the intracellular anti-inflammatory mechanism of IL-37. It also confirms that IL-37 decreases with cellular senescence and that increasing intracellular IL-37 can effectively treat synovitis and synovitis-induced inflammatory damage to cartilage.

Alpha-mangostin alleviate renal interstitial fibrosis via suppression of TGF-β1/Smad/ERK signaling axis in vitro and in vivo

α-mangostin (α-MG), a natural derivative of coumarin, exhibits anti-inflammatory, antioxidant and anti-fibrotic effects. This study aimed to determine the effect of α-MG treatment in mediating the process of renal interstitial fibrosis. We found that α-MG could alleviate tubule-interstitial damage and decrease fibrotic (α-smooth muscle actin [α-SMA], fibronectin, and collagen I), and epithelial-mesenchymal transition (EMT) protein (N-cadherin, Snail, Slug, TGF-β1 and vimentin) expression in unilateral ureteral obstruction (UUO) mice with chronic kidney disease. α-MG significantly decreased motility as well as inhibited expression of fibrotic- and EMT-related proteins in TGF-β1-induced HK2 cells. To clarify the molecular mechanisms of α-MG in reducing renal interstitial fibrosis, we used a MEK inhibitor (U0126) or Smad inhibitor (SB431542) cotreatment with α-MG. This is the first study is to demonstrate the antifibrotic effects of α-MG by targeting the TGF-β1/ERK/Smad-mediated EMT signaling pathway, is even more effective against renal interstitial fibrosis.

Abstract 19030: Growth Arrest-Specific 5, a Novel Regulator of Adipose Tissue Browning

Background: Obesity is a global epidemic with significant health risks, including type 2 diabetes and cardiovascular diseases. Increasing white adipose tissue (WAT) browning has shown metabolic benefits in both animal models and humans. Long non-coding RNAs (lncRNAs), have been implicated in various diseases, including metabolic disorders. Growth arrest-specific 5 (GAS5), a tumor suppressor lncRNA decreased in type 2 diabetic patients, has been found to be involved in adipocyte differentiation, however, the role of GAS5 in WAT browning has not yet been established. We hypothesized that in the presence of an adipose tissue browning stimuli, GAS5 promotes WAT browning Methods: In vitro approaches through knocking down or overexpressing GAS5 were used to determine its role in regulating thermogenic genes and thermogenesis. To test the role of GAS5 in adipose tissue browning in vivo , adenovirus AAV8 expressing GAS5 was administered specifically in epidydimal adipose tissues followed by the treatment of browning stimuli CL-316,243. Results: Knockdown of GAS5 increased Smad3 phosphorylation, which was blocked by Smad3 inhibitor, SIS3. Conversely, overexpression of GAS5 inhibited Smad3 phosphorylation. On the other hand, Smad3 overexpression blocked the stimulatory effects of GAS5 on the expression of thermogenic genes PGC-1α and UCP1. Moreover, overexpression of GAS5 inhibited, while knockdown of GAS5 enhanced, Smad3 nuclear translocation and tis binding to PGC-1α promoter. In vivo experiments with GAS5 overexpression in epididymal adipose tissues revealed that GAS5 promotes CL-316,243-induced WAT browning in mice. Conclusions: Our study demonstrates that GAS5 promotes WAT browning by suppressing Smad3 signaling, which in turn increases thermogenesis via stimulating PGC-1α and UCP1 expression. These findings establish GAS5 as a novel regulator of WAT browning with potential implications for mitigating obesity and associated metabolic disorders.

Rewiring of the promoter-enhancer interactome and regulatory landscape in glioblastoma orchestrates gene expression underlying neurogliomal synaptic communication

Chromatin organization controls transcription by modulating 3D-interactions between enhancers and promoters in the nucleus. Alterations in epigenetic states and 3D-chromatin organization result in gene expression changes contributing to cancer. Here, we map the promoter-enhancer interactome and regulatory landscape of glioblastoma, the most aggressive primary brain tumour. Our data reveals profound rewiring of promoter-enhancer interactions, chromatin accessibility and redistribution of histone marks in glioblastoma. This leads to loss of long-range regulatory interactions and overall activation of promoters, which orchestrate changes in the expression of genes associated to glutamatergic synapses, axon guidance, axonogenesis and chromatin remodelling. SMAD3 and PITX1 emerge as major transcription factors controlling genes related to synapse organization and axon guidance. Inhibition of SMAD3 and neuronal activity stimulation cooperate to promote proliferation of glioblastoma cells in co-culture with glutamatergic neurons, and in mice bearing patient-derived xenografts. Our findings provide mechanistic insight into the regulatory networks that mediate neurogliomal synaptic communication.