Molecular basis of CX-5461-induced DNA damage response in primary vascular smooth muscle cells.

Our previous studies have shown that the novel selective RNA polymerase I inhibitor CX-5461 suppresses proliferation of vascular smooth muscle cells, mainly by inducing DNA damage response (DDR), including activations of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) and p53. Currently, there is no information about the molecular mechanism(s) underlying CX-5461-induced DDR in vascular cells, while the results obtained in cancer cells and immortalized cell lines are controversial. In this study, we examined the responses of various DDR pathways to CX-5461 treatment in primary aortic smooth muscle cells isolated from normal adult Sprague Dawley rats. We demonstrated that CX-5461-induced DDR was not associated with activations of the nucleotide excision repair, DNA mismatch repair, or the non-homologous end joining pathways, while the homologous recombination pathway was activated. However, the alkaline comet assay did not show massive DNA double strand breaks in CX-5461-treated cells. Instead, CX-5461-induced DDR appeared to be related to induction of DNA replication stress, which was not attributable to increased formation of G-quadruplex or R-loop structures, but might be explained by the increased replication-transcription conflict. CX-5461-induced DDR was not exclusively confined to rDNA within the nucleolar compartment; the extra-nucleolar DDR might represent a distinct secondary response related to the downregulated Rad51 expression in CX-5461-treated cells. In summary, we suggest that DNA replication stress may be the primary molecular event leading to downstream ATM/ATR and p53 activations in CX-5461-treated vascular smooth muscle cells. Our results provide further insights into the molecular basis of the beneficial effects of CX-5461 in proliferative vascular diseases.

The management pattern and outcomes of chronic thromboembolic pulmonary hypertension: rationale and design for a Chinese real-world study.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive pulmonary vascular disorder with substantial morbidity and mortality, also a disease underdiagnosed and undertreated. It is potentially curable by pulmonary endarterectomy (PEA) in patients with surgically accessible thrombi. Balloon pulmonary angioplasty (BPA) and targeted medical therapy are options for patients with distal lesions or persistent/recurrent pulmonary hypertension after PEA. There is an urgent need to increase the awareness of CTEPH. Qualified CTEPH centers are still quite limited. Baseline characteristics, management pattern and clinical outcome of CTEPH in China needs to be reported. METHODS AND DESIGN: The CHinese reAl-world study to iNvestigate the manaGEment pattern and outcomes of chronic thromboembolic pulmonary hypertension (CHANGE) study is designed to provide the multimodality treatment pattern and clinical outcomes of CTEPH in China. Consecutive patients who are ≥ 14 year-old and diagnosed with CTEPH are enrolled. The diagnosis of CTEPH is confirmed in right heart catheterization and imaging examinations. The multimodality therapeutic strategy, which consists of PEA, BPA and targeted medical therapy, is made by a multidisciplinary team. The blood sample and tissue from PEA are stored in the central biobank for further research. The patients receive regular follow-up every 3 or 6 months for at least 3 years. The primary outcomes include all-cause mortality and changes in functional and hemodynamic parameters from baseline. The secondary outcomes include the proportion of patients experiencing lung transplantation, the proportion of patients experiencing heart and lung transplantation, and changes in health-related quality of life. Up to 31 December 2023, the study has enrolled 1500 eligible patients from 18 expert centers. CONCLUSIONS: As a real-world study, the CHANGE study is expected to increase our understanding of CTEPH, and to fill the gap between guidelines and the clinical practice in the diagnosis, assessment and treatment of patients with CTEPH. REGISTRATION NUMBER IN CLINICALTRIALS.GOV: NCT05311072.

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.

Optimal combination of right ventricular functional parameters using echocardiography in pulmonary arterial hypertension.

AIMS: Novel echocardiographic parameters of right ventricular (RV) function, including speckle-tracking-derived, three-dimensional, and RV-pulmonary artery coupling parameters, have emerged for the evaluation of pulmonary arterial hypertension (PAH). The relative role of these parameters in the risk stratification of PAH patients is unclear. We compared the performance of multiple RV parameters and sought to establish an optimal model for identifying the risk profile of patients with PAH. METHODS AND RESULTS: Comprehensive risk assessments were performed for 70 patients with PAH. The risk profile of every patient was determined based on the guideline recommendations. Conventional parameters, including fractional area change (FAC) and tricuspid annular plane systolic excursion (TAPSE), novel speckle-tracking-derived RV longitudinal strain (RVLS), and three-dimensional RV ejection fraction (3D-RVEF), were used to evaluate RV function. Pressure-strain loops were measured for the assessment of RV myocardial work, including RV global wasted work (RVGWW). RV-pulmonary artery coupling was assessed by indexing RV parameters to the estimated pulmonary artery systolic pressure (PASP). The median age was 34 (30-43) years, and 62 (88.6%) patients were female. Forty-five patients were classified into the low-risk group, while 25 patients were classified into the intermediate-high-risk group. Most RV parameters could be used to determine the risk profile and exhibited significantly improved diagnostic performance after indexing to PASP (including FAC/PASP, TAPSE/PASP, and 3D-RVEF/PASP). RVLS/PASP showed the best performance, with an area under the curve of 0.895. In multivariate analysis (Model 1), only RVGWW (>90.5 mmHg%), RVLS (> -16.7%), and TAPSE (<17.5 mm) remained significant (all P < 0.05). Model 1 outperformed every single RV parameter, with a significantly larger area under the curve (all P < 0.05). With PASP indexing in Model 2, RVLS/PASP > -0.275 [odds ratio (OR) 20.63, 95% confidence interval (CI) 4.62-92.11, P < 0.001] and RVGWW > 90.5 mmHg% (OR 6.17, 95% CI 1.37-27.76, P = 0.018) independently identified a higher risk profile. The addition of RVGWW to two models determined incremental value in identification (continuous net reclassification improvement 1.058, 95% CI 0.639-1.477, P < 0.001). CONCLUSIONS: The combination models for RV function outperformed any single parameter in identifying the risk profile of patients with PAH. Comprehensive assessment of RV-pulmonary artery coupling using multiparametric methods is clinically meaningful in patients with PAH.

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.

Boosting regulatory T cell-dependent immune tolerance by activation of p53.

Regulatory T cells (Tregs) have critical roles in maintaining immune hemostasis and have important anti-inflammatory functions in diseases. Recently, we identified that CX-5461 (a selective RNA polymerase I inhibitor and p53 activator) acted as a potent immunosuppressive agent, which prevented allogeneic acute rejection in animal models via a molecular mechanism distinct from all those of conventional immunosuppressive drugs. Unexpectedly, we discovered that CX-5461 could promote Treg differentiation. In this review, we have summarized the evidence for a potential role of p53 in mediating Treg differentiation and its possible mechanisms, including regulation of FoxP3 transcription, regulation of the expression of PTEN (phosphatase and tensin homolog), as well as protein-protein interaction with the transcription factor STAT5 (signal transducer and activator of transcription 5). Evidence also suggests that pharmacological p53 activators may potentially be used to boost Treg-mediated immune tolerance. Based on these data, we argue that novel p53 activators such as CX-5461 may represent a distinct class of immunosuppressants that repress conventional T cell-mediated alloimmunity with concomitant boosting of Treg-dependent immune tolerance.

Ferroptosis-related lncRNA model based on CFAP58-DT for predicting prognosis and immunocytes infiltration in endometrial cancer.

Background: Endometrial cancer (EC) is a kind of common gynecological tumor. Further study on the markers related to the prognosis of endometrial cancer is important for women worldwide. Methods: The Cancer Genome Atlas (TCGA) database was used to obtain the transcriptome profiling and clinical data. A model was built using packages based on R software. Immune-related databases were employed to analyze the infiltration of immunocytes. Quantitative real-time PCR (qRT-PCR), cell counting kit-8 (CCK-8), and transwell assays were utilized to investigate the role of CFAP58-DT in EC. Results: Following Cox regression analysis, 1,731 ferroptosis-related long non-coding RNA (lncRNA) were screened, and a 9-related lncRNA prognostic model was constructed. Patients were classified as high- and low-risk according to their expression spectrum. Kaplan-Meier (KM) analysis showed that the prognosis of low-risk patients was poor. Operating characteristic curves, decision curve analysis, and a nomogram suggested the model could independently guide prognostic evaluation, with higher sensitivity, specificity, and efficiency than other common clinical characteristics. Gene Set Enrichment Analysis (GSEA) was conducted to determine the enriched pathways among the two groups and evaluation of the immune-infiltrating conditions were performed to help improve immune therapy. Finally, we conducted cytological studies on the model's most important indicators. Conclusions: Overall, we identified a prognostic ferroptosis-related lncRNA model based on CFAP58-DT for predicting the prognosis and immune-infiltrating conditions in EC. We concluded that the potential oncogenic role of CFAP58-DT can further guide immunotherapy and chemotherapy.

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.

Selexipag-based triple combination therapy improves prognosis in Chinese pulmonary arterial hypertension patients.

Aim: Selexipag is an oral selective prostacyclin receptor agonist approved for treatment of patients with pulmonary arterial hypertension (PAH). In the present study, we aim to assess the safety and efficacy of selexipag in triple combination therapy with endothelial receptor antagonists (ERAs) and PDE5is for Chinese PAH patients. Methods and results: A single center retrospective study was performed on group 1 PAH patients (n = 68) initiating triple combination therapy with selexipag from 1 February 2020 to 31 August 2021 in Qilu Hospital of Shandong University (Shandong, China). Adolescents, children, and PAH patients with unrepaired congenital heart disease were excluded. The French pulmonary hypertension network (FPHN) non-invasive risk assessment, echocardiogram parameters, and clinical data, including tolerability, safety, and death/hospitalization events associated with PAH, were collected. Of the 68 patients, 31 (45.6%) patients had tolerable side effects while only a single patient discontinued selexipag due to severe diarrhea. In the analysis of the efficacy set of 62 patients, the median selexipag treatment time from selexipag initiation to last risk assessment was 27 (21, 33) weeks. Compared to baseline parameters, the percentage of WHO FC III/IV decreased from 77.4% (48) to 24.2% (15) (p = 0.000), median 6-min walk distance (6MWD) increased 82 m [from 398 (318, 450) to 480 (420, 506) m; p = 0.000], and NT-proBNP levels decreased from 1,216 (329, 2,159) to 455 (134, 1,678) pg/mL (p = 0.007). Patients who improved to three low-risk criteria increased from 9.7 to 38.7%. Right ventricular diameter (RV) diameter also decreased and was accompanied by an improved tricuspid annular plane systolic excursion (TAPSE). Patients transitioning from subcutaneous treprostinil to selexipag continued to show improvements in WHO FC, 6MWD (404 ± 94 vs. 383 ± 127 m) and NT-proBNP levels (2,319 ± 2,448 vs. 2,987 ± 3,770 pg/mL). Finally, the 1-year event free survival rate was 96.7% for patients initiating the triple combination therapy within 3 years of PAH diagnosis. Conclusion: Triple combination therapy with selexipag was safe and effective in Chinese PAH patients, which was confirmed by acceptable tolerability, and improved exercise capacity, right heart function, risk assessment, and prognosis.

S100A9 promotes inflammatory response in diabetic nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has been previously shown to be associated with diabetes mellitus (DM) which is one of the most decisive risk factors for the faster progression of NAFLD to nonalcoholic steatohepatitis (NASH), fibrosis or advanced cirrhosis. However, the critical molecular pathway involved in the development of diabetic-induced liver injury is unclear. By the proteomic study of liver from high-fat diet (HFD)/streptozotocin(STZ)-induced diabetic mice, we revealed that the upregulation of S100A9 was involved in the development of NAFLD with DM. Moreover, we found that S100A9 silencing decreased proinflammatory response and inhibited the TLR4-NF-κB signaling in in-vitro study. Our findings provide new perspectives into the pivotal role of S100A9 for development of diabetic NAFLD and revealed that S100A9 is a critical molecule that links liver injury to inflammation of NAFLD with DM.

Phloretin ameliorates diabetic nephropathy by inhibiting nephrin and podocin reduction through a non-hypoglycemic effect.

Phloretin is a dihydrochalcone flavonoid from natural plants, which has protective activities against oxidative stress and inflammation. To date, its effect on diabetic nephropathy (DN) has not been investigated. In this study, we examined the potential role of phloretin in diabetes-induced renal damage and associated mechanisms in a type 2 diabetes mellitus (T2DM) model induced by streptozotocin (STZ) and high-fat diet (HFD) in Apolipoprotein E knockout (ApoE-/-) mice. We found that daily treatment with a low dose (20 mg kg-1) of phloretin, as a dietary supplement, significantly alleviated polyuria, proteinuria, and glomerular histopathological changes in the T2DM mice, indicating a protective effect of phloretin on diabetic renal dysfunction. In the phloretin-treated T2DM mice, major metabolic parameters, including blood glucose levels, were not altered significantly, suggesting that the observed beneficial effects of phloretin may be due to a mechanism independent of blood glucose control. Further experiments revealed that phloretin had a protective effect on glomerular podocytes as indicated by ameliorated glomerular basement membrane (GBM) thickening and podocyte foot process effacement. Moreover, phloretin treatment restored levels of nephrin and podocin, two podocyte slit diaphragm proteins that were decreased in T2DM mice. Our results indicate that low-dose phloretin treatment has a protective effect on podocytes in DN via a non-hypoglycemic mechanism in preserving nephrin and podocin expression levels. These data suggest that phloretin may be exploited as a novel therapeutic agent for DN.

CX-5461 is a potent immunosuppressant which inhibits T cell-mediated alloimmunity via p53-DUSP5.

CX-5461 is a first-in-class selective RNA polymerase I inhibitor. Previously we found that CX-5461 had anti-inflammatory activities. In this study we characterized potential immunosuppressive effects of CX-5461 and explored the underlying mechanisms. Allogeneic skin transplantation model (BALB/c to C57BL/6 mice) and heterotopic heart transplantation model (F344 to Lewis rats) were used. We showed that CX-5461 was a potent inhibitor of alloimmunity which prevented acute allograft rejections. CX-5461 treatment was invariably associated with expansion of the regulatory T cell population. In vitro, CX-5461 inhibited agonists-induced T cell activation. CX-5461 consistently inhibited the expression of interferon-γ and interleukin - 2, key mediators of T cell-mediated alloimmunity. Mechanistically, CX-5461-induced immunosuppression was, at least partly, dependent on the p53-DUSP5 (dual-specificity phosphatase 5) axis and subsequent antagonism of the Erk1/2 mitogen-activated protein kinase pathway. In conclusion, our results suggest that CX-5461 is a promising candidate of a novel class of immunosuppressant which may be used as an alternative to the currently approved anti-rejection therapies.

Structural and functional insights into a novel pre-clinical-stage antibody targeting IL-17A for treatment of autoimmune diseases.

The pro-inflammatory cytokine interleukin-17A (IL-17A) is a key driver of multiple inflammatory and immune disorders. Therapeutic antibodies targeting IL-17A have been proven effective in treating patients with these diseases; however, large variations in clinical outcomes have been observed with different antibodies. In this study, we developed HB0017, a novel monoclonal antibody that targets human IL-17A. HB0017 specifically and strongly bound to human, cynomolgus monkey, and mouse IL-17A at the physiological interface with the IL-17A receptor. In human and monkey cells, HB0017 potently antagonized the functions of IL-17A through competitive binding. HB0017 functioned equivalently to that of clinically approved antibodies in terms of therapeutic efficacy for inflammatory disorders and psoriasis in a mouse model. The results indicate that HB0017 may be an alternative biological therapy for treating patients with inflammation and autoimmune diseases.

Amino acid starvation-induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance.

Mammalian cells utilize Akt-dependent signaling to deploy intracellular Glut4 toward cell surface to facilitate glucose uptake. Low-density lipoprotein receptor (LDLR) is the cargo receptor mediating endocytosis of apolipoprotein B-containing lipoproteins. However, signaling-controlled regulation of intracellular LDLR trafficking remains elusive. Here, we describe a unique amino acid stress response, which directs the deployment of intracellular LDLRs, causing enhanced LDL endocytosis, likely via Ca2+ and calcium/calmodulin-dependent protein kinase II-mediated signalings. This response is independent of induction of autophagy. Amino acid stress-induced increase in LDL uptake in vitro is comparable to that by pravastatin. In vivo, acute AAS challenge for up to 72 h enhanced the rate of hepatic LDL uptake without changing the total expression level of LDLR. Reducing dietary amino acids by 50% for 2 to 4 weeks ameliorated high fat diet-induced hypercholesterolemia in heterozygous LDLR-deficient mice, with reductions in both LDL and VLDL fractions. We suggest that identification of signaling-controlled regulation of intracellular LDLR trafficking has advanced our understanding of the LDLR biology, and may benefit future development of additional therapeutic strategies for treating hypercholesterolemia.

A Novel Bispecific Antibody Targeting PD-L1 and VEGF With Combined Anti-Tumor Activities.

Therapeutic monoclonal antibodies (mAbs) blocking immune checkpoints have been mainly used as monotherapy. Recently, combination therapy targeting multiple immune checkpoints has recently been explored to increase anti-cancer efficacy. Particularly, a single molecule targeting more than one checkpoints has been investigated. As dual blocking of PD-1/PD-L1 and VEGF/VEGFR has demonstrated synergism in anti-tumor activities, we developed a novel bispecific antibody, termed HB0025, which is formed via fusing the domain 2 of vascular endothelial growth factor receptor 1 (VEGFR1D2) and anti-PD-L1 mAb by using mAb-Trap technology. HB0025 almost completely retains the binding affinities and the biological activities in-vitro when compared with the parent anti-PD-L1 mAb or VEGFR1D2 fusion protein. Preclinical data demonstrated that HB0025 was more effective in inhibiting cancer growth than anti PD-L1 mAb or VEGFR1D2 fusion protein. Thus, our bispecific antibody may bring about greater clinical benefits and broader indications.

Combined transcriptomic and lipidomic analysis of D-4F ameliorating bleomycin-induced pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease that leads to respiratory failure, and for which there is no effective treatment. Apolipoprotein A-1 (ApoA-1) has been reported to ameliorate the bleomycin (BLM)-induced IPF model. METHODS: To examine the function of D-4F, an ApoA-1 mimetic polypeptide, in IPF, we used an in-vivo BLM-induced model. We assigned mice into the following 3 groups: the Blank Group (BLK Group), the Bleomycin Treatment Group (Model Group), and the D-4F Interference Group (Inter Group). The BLM-induced fibrosis was examined by hematoxylin and eosin, Masson's trichrome (M-T) staining and immunohistochemical staining. An untargeted lipidomic and transcriptomic analysis were used to examine the function of D-4F. RESULTS: There were 35 differentially altered lipids (DALs) in the BLK, Model and Inter Groups. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that glycerophospholipid metabolism was the most highly enriched of the 35 DALs. There were 99 differentially expressed genes (DEGs) in the BLK, Model and Inter Groups. The enriched KEGG pathway analysis showed that the mitogen-activated protein kinase (MAPK) pathway was 1 of the top 10 pathways. The results of the untargeted lipidomic and transcriptomic analysis showed that phospholipase A2 group 4c (Pla2g4c) was a crucial gene in both the MAPK pathway and glycerophospholipid metabolism. Pla2g4c was increased in the Model Group but decreased in the Inter Group. CONCLUSIONS: It may be that D-4F prevented the BLM-induced pulmonary fibrosis model by inhibiting the expression of pla2g4c. Our findings suggest that D-4F may be a potential treatment of IPF.

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.

The p53 pathway in vasculature revisited: A therapeutic target for pathological vascular remodeling?

Pathological vascular remodeling contributes to the development of restenosis following intraluminal interventions, transplant vasculopathy, and pulmonary arterial hypertension. Activation of the tumor suppressor p53 may counteract vascular remodeling by inhibiting aberrant proliferation of vascular smooth muscle cells and repressing vascular inflammation. In particular, the development of different lines of small-molecule p53 activators ignites the hope of treating remodeling-associated vascular diseases by targeting p53 pharmacologically. In this review, we discuss the relationships between p53 and pathological vascular remodeling, and summarize current experimental data suggesting that drugging the p53 pathway may represent a novel strategy to prevent the development of vascular remodeling.

Ginsenoside Rb3 Alleviates CSE-induced TROP2 Upregulation through p38 MAPK and NF-κB Pathways in Basal Cells.

Smoking-mediated reprogramming of the phenotype and function of airway basal cells (BCs) disrupts airway homeostasis and is an early event in chronic obstructive pulmonary disease (COPD)-associated airway remodeling. Here, we examined the expression and regulation of the transmembrane glycoprotein TROP2 (trophoblast antigen 2), a putative stem cell marker in airway BCs, in lung tissue samples from healthy smokers and healthy nonsmokers and in models in culture to identify therapeutic targets. TROP2 expression was upregulated in the airway epithelia of smokers and positively correlated with the smoking index. In vitro, cigarette smoke extract (CSE) induced TROP2 expression in airway BCs in a time- and dose-dependent manner. The p38 MAPK and NF-κB pathways were also activated by CSE, and their specific antagonists inhibited CSE-induced TROP2 expression. A therapeutic component derived from traditional Chinese medicine, ginsenoside Rb3, inhibited CSE-induced TROP2 expression as well as activation of the p38 MAPK and NF-κB pathways in BCs in monolayer culture. Furthermore, ginsenoside Rb3 prevented the increase in TROP2 expression and antagonized CSE-induced BC hyperplasia and expression of inflammatory factors and epithelial-mesenchymal transition changes in an air-liquid culture model. Thus, CSE-induced TROP2 is a possible biomarker for early changes in the epithelium of smokers, and ginsenoside Rb3 may serve as a therapeutic molecule, preventing the disruption of epithelial homeostasis in COPD.

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.