Identification of a distinct cluster of GDF15high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities.

Introduction: Macrophage-mediated inflammatory response may have crucial roles in the pathogenesis of a variety of human diseases. Growth differentiation factor 15 (GDF15) is a cytokine of the transforming growth factor-ß superfamily, with potential anti-inflammatory activities. Previous studies observed in human lungs some macrophages which expressed a high level of GDF15. Methods: In the present study, we employed multiple techniques, including immunofluorescence, flow cytometry, and single-cell RNA sequencing, in order to further clarify the identity of such GDF15high macrophages. Results: We demonstrated that macrophages derived from human peripheral blood mononuclear cells and rat bone marrow mononuclear cells by in vitro differentiation with granulocyte-macrophage colony stimulating factor contained a minor population (~1%) of GDF15high cells. GDF15high macrophages did not exhibit a typical M1 or M2 phenotype, but had a unique molecular signature as revealed by single-cell RNA sequencing. Functionally, the in vitro derived GDF15high macrophages were associated with reduced responsiveness to pro-inflammatory activation; furthermore, these GDF15high macrophages could inhibit the pro-inflammatory functions of other macrophages via a paracrine mechanism. We further confirmed that GDF15 per se was a key mediator of the anti-inflammatory effects of GDF15high macrophage. Also, we provided evidence showing that GDF15high macrophages were present in other macrophage-residing human tissues in addition to the lungs. Further scRNA-seq analysis in rat lung macrophages confirmed the presence of a GDF15high sub-population. However, these data indicated that GDF15high macrophages in the body were not a uniform population based on their molecular signatures. More importantly, as compared to the in vitro derived GDF15high macrophage, whether the tissue resident GDF15high counterpart is also associated with anti-inflammatory functions remains to be determined. We cannot exclude the possibility that the in vitro priming/induction protocol used in our study has a determinant role in inducing the anti-inflammatory phenotype in the resulting GDF15high macrophage cells. Conclusion: In summary, our results suggest that the GDF15high macrophage cells obtained by in vitro induction may represent a distinct cluster with intrinsic anti-inflammatory functions. The (patho)physiological importance of these cells in vivo warrants further investigation.

The regulation of MFG-E8 on the mitophagy in diabetic sarcopenia via the HSPA1L-Parkin pathway and the effect of D-pinitol.

BACKGROUND: Diabetic sarcopenia is a disease-related skeletal muscle disorder that causes progressive symptoms. The complete understanding of its pathogenesis is yet to be unravelled, which makes it difficult to develop effective therapeutic strategies. This study investigates how MFG-E8 affects mitophagy and the protective role of D-pinitol (DP) in diabetic sarcopenia. METHODS: In vivo, streptozotocin-induced diabetic SAM-R1 (STZ-R1) and SAM-P8 (STZ-P8) mice (16-week-old) were used, and STZ-P8 mice were administrated of DP (150 mg/kg per day) for 6 weeks. Gastrocnemius muscles were harvested for histological analysis including transmission electron microscopy. Proteins were evaluated via immunohistochemistry (IHC), immunofluorescence (IF), and western blotting (WB) assay. In vitro, advanced glycation end products (AGEs) induced diabetic and D-galactose (DG) induced senescent C2C12 models were established and received DP, MFG-E8 plasmid (Mover)/siRNA (MsiRNA), or 3-MA/Torin-1 intervention. Proteins were evaluated by IF and WB assay. Immunoprecipitation (IP) and co-immunoprecipitation (CO-IP) were used for hunting the interacted proteins of MFG-E8. RESULTS: In vivo, sarcopenia, mitophagy deficiency, and up-regulated MFG-E8 were confirmed in the STZ-P8 group. DP exerted protective effects on sarcopenia and mitophagy (DP + STZ-P8 vs. STZ-P8; all P < 0.01), such as increased lean mass (8.47 ± 0.81 g vs. 7.08 ± 1.64 g), grip strength (208.62 ± 39.45 g vs. 160.87 ± 26.95 g), rotarod tests (109.7 ± 11.81 s vs. 59.3 ± 20.97 s), muscle cross-sectional area (CSA) (1912.17 ± 535.61 µm2 vs. 1557.19 ± 588.38 µm2), autophagosomes (0.07 ± 0.02 per µm2 vs. 0.02 ± 0.01 per µm2), and cytolysosome (0.07 ± 0.03 per µm2 vs. 0.03 ± 0.01 per µm2). DP down-regulated MFG-E8 in both serum (DP + STZ-P8: 253.19 ± 34.75 pg/mL vs. STZ-P8: 404.69 ± 78.97 pg/mL; P < 0.001) and gastrocnemius muscle (WB assay. DP + STZ-P8: 0.39 ± 0.04 vs. STZ-P8: 0.55 ± 0.08; P < 0.01). DP also up-regulated PINK1, Parkin and LC3B-II/I ratio, and down-regulated P62 in gastrocnemius muscles (all P < 0.01). In vitro, mitophagy deficiency and MFG-E8 up-regulation were confirmed in diabetic and senescent models (all P < 0.05). DP and MsiRNA down-regulated MFG-E8 and P62, and up-regulated PINK1, Parkin and LC3B-II/I ratio to promote mitophagy as Torin-1 does (all P < 0.05). HSPA1L was confirmed as an interacted protein of MFG-E8 in IP and CO-IP assay. Mover down-regulated the expression of Parkin via the HSPA1L-Parkin pathway, leading to mitophagy inhibition. MsiRNA up-regulated the expression of PINK1 via SGK1, FOXO1, and STAT3 phosphorylation pathways, leading to mitophagy stimulation. CONCLUSIONS: MFG-E8 is a crucial target protein of DP and plays a distinct role in mitophagy regulation. DP down-regulates the expression of MFG-E8, reduces mitophagy deficiency, and alleviates the symptoms of diabetic sarcopenia, which could be considered a novel therapeutic strategy for diabetic sarcopenia.

CX­5461 potentiates imatinib­induced apoptosis in K562 cells by stimulating KIF1B expression.

The selective RNA polymerase I inhibitor CX-5461 has been shown to be effective in treating some types of leukemic disorders. Emerging evidence suggests that combined treatments with CX-5461 and other chemotherapeutic agents may achieve enhanced effectiveness as compared with monotherapies. Currently, pharmacodynamic properties of the combination of CX-5461 with tyrosine kinase inhibitors remain to be explored. The present study tested whether CX-5461 could potentiate the effect of imatinib in the human chronic myeloid leukemia cell line K562, which is p53-deficient. It was demonstrated that CX-5461 at 100 nM, which was non-cytotoxic in K562 cells, potentiated the pro-apoptotic effect of imatinib. Mechanistically, the present study identified that the upregulated expression of kinesin family member 1B (KIF1B) gene might be involved in mediating the pro-apoptotic effect of imatinib/CX-5461 combination. Under the present experimental settings, however, neither CX-5461 nor imatinib alone exhibited a significant effect on KIF1B expression. Moreover, using other leukemic cell lines, it was demonstrated that regulation of KIF1B expression by imatinib/CX-5461 was not a ubiquitous phenomenon in leukemic cells and should be studied in a cell type-specific manner. In conclusion, the results suggested that the synergistic interaction between CX-5461 and imatinib may be of potential clinical value for the treatment of tyrosine kinase inhibitor-resistant chronic myeloid leukemia.

D-pinitol alleviates diabetic cardiomyopathy by inhibiting the optineurin-mediated endoplasmic reticulum stress and glycophagy signaling pathway.

Diabetic cardiomyopathy (DCM) is an important complication resulting in heart failure and death of diabetic patients. However, there is no effective drug for treatments. This study investigated the effect of D-pinitol (DP) on cardiac injury using diabetic mice and glycosylation injury of cardiomyocytes and its molecular mechanisms. We established the streptozotocin-induced SAMR1 and SAMP8 mice and DP (150 mg/kg/day) intragastrically and advanced glycation end-products (AGEs)-induced H9C2 cells. H9C2 cells were transfected with optineurin (OPTN) siRNA and overexpression plasmids. The metabolic disorder indices, cardiac dysfunction, histopathology, immunofluorescence, western blot, and immunoprecipitation were investigated. Our results showed that DP reduced the blood glucose and AGEs, and increased the expression of heart OPTN in diabetic mice and H9C2 cells, thereby inhibiting the endoplasmic reticulum stress (GRP78, CHOP) and glycophagy (STBD1, GABARAPL1), and alleviating the myocardial apoptosis and fibrosis of DCM. The expression of filamin A as an interaction protein of OPTN downregulated by AGEs decreased OPTN abundance. Moreover, OPTN siRNA increased the expression of GRP78, CHOP, STBD1, and GABARAPL1 and inhibited the expression of GAA via GSK3ß phosphorylation and FoxO1. DP may be helpful to treat the onset of DCM. Targeting OPTN with DP could be translated into clinical application in the fighting against DCM.

Efficiency and impact factors of anatomical intelligence for breast and hand-held ultrasound in lesion detection.

PURPOSE: To investigate the efficiency and impact factors of anatomical intelligence for breast (AI-Breast) and hand-held ultrasound (HHUS) in lesion detection. METHODS: A total of 172 outpatient women were randomly selected, underwent AI-Breast ultrasound (Group AI) once and HHUS twice. HHUS was performed by breast imaging radiologists (Group A) and general radiologists (Group B). For the AI-Breast examination, a trained technician performed the whole-breast scan and data acquisition, while other general radiologists performed image interpretation. The examination time and lesion detection rate were recorded. The impact factors for breast lesion detection, including breast cup size, number of lesions, and benign or malignant lesions were analyzed. RESULTS: The detection rates of Group AI, A, and B were 92.8 ± 17.0%, 95.0 ± 13.6%, and 85.0 ± 22.9%, respectively. Comparable lesion detection rates were observed in Group AI and Group A (P > 0.05), but a significantly lower lesion detection rate was observed in Group B compared to the other two (both P < 0.05). Regarding missed diagnosis rates of malignant lesions, comparable performance was observed in Group AI, Group A, and Group B (8% vs. 4% vs. 14%, all P > 0.05). Scan times of Groups AI, A, and B were 262.15 ± 40.4 s, 237.5 ± 110.3 s, 281.2 ± 86.1 s, respectively. The scan time of Group AI was significantly higher than Group A (P < 0.01), but was slightly lower than Group B (P > 0.05). We found a strong linear correlation between scan time and cup size in Group AI (r = 0.745). No impacts of cup size and number of lesions were found on the lesion detection rate in Group AI (P > 0.05). CONCLUSIONS: With the assist of AI-Breast system, the lesion detection rate of AI-Breast ultrasound was comparable to that of a breast imaging radiologist and superior to that of the general radiologist. AI-Breast ultrasound may be used as a potential approach for breast lesions surveillance.

Novel RNA polymerase I inhibitor CX-5461 suppresses imiquimod-induced experimental psoriasis.

Clinical treatment of psoriasis remains challenging because of possible long-term drug toxicities and loss of therapeutic effects over time. CX-5461 is a novel selective inhibitor of RNA polymerase I. Our previous studies have shown that CX-5461 has potent anti-inflammatory effects. Here we investigated whether CX-5461 could inhibit the development of imiquimod-induced experimental psoriasis in mice. Adult male C57BL/6 mice were used, and psoriasis-like lesions were induced by topical imiquimod treatment. In vivo, we demonstrated that topical application of CX-5461 prevented the development of imiquimod-induced psoriasis, with decreases in keratinocyte proliferation, T-cell infiltration and pathological angiogenesis. CX-5461 also reversed existing skin inflammation induced imiquimod and retarded the development of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and inflammation. In vitro, CX-5461 induced cell cycle arrest in keratinocytes, inhibited expressions of interleukin-17, interleukin-23 receptor and retinoic acid receptor-related orphan receptor-γt in activated T cells, and reduced angiogenic functions of endothelial cells. In conclusion, CX-5461 exhibits therapeutic effects on experimental psoriasis in mice, likely via multiple mechanisms including anti-proliferative, anti-inflammatory and anti-angiogenic activities.

A transcriptional program associated with cell cycle regulation predominates in the anti-inflammatory effects of CX-5461 in macrophage.

CX-5461, a novel selective RNA polymerase I inhibitor, shows potential anti-inflammatory and immunosuppressive activities. However, the molecular mechanisms underlying the inhibitory effects of CX-5461 on macrophage-mediated inflammation remain to be clarified. In the present study, we attempted to identify the systemic biological processes which were modulated by CX-5461 in inflammatory macrophages. Primary peritoneal macrophages were isolated from normal Sprague Dawley rats, and primed with lipopolysaccharide or interferon-γ. Genome-wide RNA sequencing was performed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used for gene functional annotations. Enrichment analysis was conducted using the ClusterProfiler package of R software. We found that CX-5461 principally induced a molecular signature related to cell cycle inhibition in primed macrophages, featuring downregulation of genes encoding cell cycle mediators and concomitant upregulation of cell cycle inhibitors. At the same concentration, however, CX-5461 did not induce a systemic anti-inflammatory transcriptional program, although some inflammatory genes such as IL-1ß and gp91phox NADPH oxidase were downregulated by CX-5461. Our data further highlighted a central role of p53 in orchestrating the molecular networks that were responsive to CX-5461 treatment. In conclusion, our study suggested that limiting cell proliferation predominated in the inhibitory effects of CX-5461 on macrophage-mediated inflammation.

The hidden breast lesions: A case report of bilateral breast cancer.

Bilateral breast cancer (BBC) is rare and is associated with an unfavorable prognosis. Consequently it is crucial to improve diagnostic performance of breast cancer in the clinical setting. We report a case of BBC in a 66-year-old woman and describe the imaging findings, including mammography, hand-held ultrasound, automated breast ultrasound, anatomical intelligence for breast ultrasound (AI-breast), and magnetic resonance imaging. Only AI-breast ultrasound successfully located the two tumors, while other imaging examinations failed to detect the tumor in the right breast.

Anti-fibrotic effects of p53 activation induced by RNA polymerase I inhibitor in primary cardiac fibroblasts.

Several lines of studies have indicated that the p53 pathway may have important anti-fibrotic functions. Previously we found that the novel selective RNA polymerase I inhibitor CX-5461 induced a robust response of p53 phosphorylation and activation in vascular smooth muscle cells. In the present study, we characterized the anti-fibrotic effects of CX-5461 in primary cardiac fibroblasts. We showed that CX-5461 suppressed spontaneous and mitogen-stimulated activation, proliferation, and myofibroblast differentiation, at a concentration (1 µM) with no cytotoxicity. The inhibitory effects of CX-5461 were primarily mediated by activation of the p53 pathway rather than limiting the rate of ribosome biogenesis. It was also shown that CX-5461 triggered a non-canonical DNA damage response in cardiac fibroblasts, which acted as the upstream signal leading to p53 activation. Taking these together, we suggest that p53 activation by pharmacological inhibition of Pol I may represent a viable approach to repress the development of cardiac fibrosis.

Therapeutic efficacy of the novel selective RNA polymerase I inhibitor CX-5461 on pulmonary arterial hypertension and associated vascular remodelling.

BACKGROUND AND PURPOSE: CX-5461 is a novel selective RNA polymerase I (Pol I) inhibitor. Previously, we found that CX-5461 could inhibit pathological arterial remodelling caused by angioplasty and transplantation. In the present study, we explored the pharmacological effects of CX-5461 on experimental pulmonary arterial hypertension (PAH) and PAH-associated vascular remodelling. EXPERIMENTAL APPROACH: PAH was induced in Sprague-Dawley rats by monocrotaline or Sugen/hypoxia. KEY RESULTS: We demonstrated that CX-5461 was well tolerated for in vivo treatments. CX-5461 prevented the development of pulmonary arterial remodelling, perivascular inflammation, pulmonary hypertension, and improved survival. More importantly, CX-5461 partly reversed established pulmonary hypertension. In vitro, CX-5461 induced cell cycle arrest in human pulmonary arterial smooth muscle cells. The beneficial effects of CX-5461 in vivo and in vitro were associated with increased activation (phosphorylation) of p53. CONCLUSION AND IMPLICATIONS: Our results suggest that pharmacological inhibition of Pol I may be a novel therapeutic strategy to treat otherwise drug-resistant PAH.

Nucleolar stress induces a senescence-like phenotype in smooth muscle cells and promotes development of vascular degeneration.

Senescence of smooth muscle cells (SMCs) has a crucial role in the pathogenesis of abdominal aortic aneurysm (AAA), a disease of vascular degeneration. Perturbation of cellular ribosomal DNA (rDNA) transcription triggers nucleolar stress response. Previously we demonstrated that induction of nucleolar stress in SMCs elicited cell cycle arrest via the ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR)-p53 axis. However, the specific roles of nucleolar stress in vascular degeneration remain unexplored. In the present study, we demonstrated for the first time that in both human and animal AAA tissues, there were non-coordinated changes in the expression of RNA polymerase I machinery components, including a downregulation of transcription initiation factor-IA (TIF-IA). Genetic deletion of TIF-IA in SMCs in mice (smTIF-IA-/-) caused spontaneous aneurysm-like lesions in the aorta. In vitro, induction of nucleolar stress triggered a non-canonical DNA damage response, leading to p53 phosphorylation and a senescence-like phenotype in SMCs. In human AAA tissues, there was increased nucleolar stress in medial cells, accompanied by localized DNA damage response within the nucleolar compartment. Our data suggest that perturbed rDNA transcription and induction of nucleolar stress contribute to the pathogenesis of AAA. Moreover, smTIF-IA-/- mice may be a novel animal model for studying spontaneous AAA-like vascular degenerations.

Pharmacological preconditioning with the cellular stress inducer thapsigargin protects against experimental sepsis.

Previous studies have shown that pretreatment with thapsigargin (TG), a cellular stress inducer, produced potent protective actions against various pathologic injuries. So far there is no information on the effects of TG on the development of bacterial sepsis. Using lipopolysaccharides- and cecal ligation/puncture-induced sepsis models in mice, we demonstrated that preconditioning with a single bolus administration of TG conferred significant improvements in survival. The beneficial effects of TG were not mediated by ER stress induction or changes in Toll-like receptor 4 signaling. In vivo and in cultured macrophages, we identified that TG reduced the protein production of pro-inflammatory cytokines, but exhibited no significant effects on steady state levels of their transcriptions. Direct measurement on the fraction of polysome-bound mRNAs revealed that TG reduced the translational efficiency of pro-inflammatory cytokines in macrophages. Moreover, we provided evidence suggesting that repression of the mTOR (the mammalian target of rapamycin) signaling pathway, but not activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-eIF2α (eukaryotic initiation factor 2α) pathway, might be involved in mediating the TG effects on cytokine production. In summary, our results support that pharmacological preconditioning with TG may represent a novel strategy to prevent sepsis-induced mortality and organ injuries.

The Selective RNA Polymerase I Inhibitor CX-5461 Mitigates Neointimal Remodeling in a Modified Model of Rat Aortic Transplantation.

BACKGROUND: Transplant vasculopathy is a major cause of chronic rejection of transplanted organs. In the present study, we examined the effects of CX-5461, a novel selective inhibitor of RNA polymerase I, on development of transplant vasculopathy using a modified model of rat aortic transplantation. METHODS: The thoracic aortas from Fischer rats were transplanted into the abdominal cavity of Lewis rats. CX-5461 was mixed in pluronic gel and administered via perivascular release. RESULTS: Treatment with CX-5461 mitigated the development of neointimal hyperplasia and vascular inflammation. This effect was likely to be attributable in part to inhibition of macrophage-dependent innate immunity reactions. Specifically, CX-5461 exhibited potent inhibitory effects on macrophage migration and lipopolysaccharide-induced activation. Treatment with CX-5461 also prevented macrophage differentiation and maturation from primary bone marrow cells. In macrophages, CX-5461 did not alter the total amount of p53 protein, but significantly increased p53 phosphorylation, which was involved in regulating cytokine-stimulated macrophage proliferation. CONCLUSIONS: In conclusion, our results suggest that pharmacological inhibition of RNA polymerase I may be a novel strategy to treat transplantation-induced arterial remodeling.

Macrophage-stimulated microRNA expression in mural cells promotes transplantation-induced neointima formation.

In this study, we tested the possibility that macrophages might contribute to neointima formation by stimulating microRNA expressions in mural cells. Thoracic aortas from F344 rats were transplanted into recipient Lewis rats. Clodronate liposome was used for in vivo macrophage depletion. Using miR-21 as a prototypic example of vascular enriched microRNA, we showed that macrophage depletion reduced the expression level of miR-21, which was upregulated in the allograft. This effect of macrophage depletion was accompanied by attenuations in neointimal hyperplasia and transplantation-induced vascular inflammation. Using in vitro assays, we identified that macrophages might stimulate miR-21 expression in smooth muscle cells and adventitial fibroblasts via the release of tumor necrosis factor-α. We also showed that silencing of miR-21 suppressed tumor necrosis factor-induced proliferation, migration, and inflammatory responses in mural cells. Our results suggest that macrophage may promote transplantation-induced neointima formation by stimulating miR-21 expression in vascular mural cells, which promotes mural cell proliferation, migration and/or inflammation. Moreover, we have established that tumor necrosis factor-α has a major role in mediating this paracrine process.