Deep neural network uncertainty estimation for early oral cancer diagnosis.

BACKGROUND: Early diagnosis in oral cancer is essential to reduce both morbidity and mortality. This study explores the use of uncertainty estimation in deep learning for early oral cancer diagnosis. METHODS: We develop a Bayesian deep learning model termed 'Probabilistic HRNet', which utilizes the ensemble MC dropout method on HRNet. Additionally, two oral lesion datasets with distinct distributions are created. We conduct a retrospective study to assess the predictive performance and uncertainty of Probabilistic HRNet across these datasets. RESULTS: Probabilistic HRNet performs optimally on the In-domain test set, achieving an F1 score of 95.3% and an AUC of 96.9% by excluding the top 30% high-uncertainty samples. For evaluations on the Domain-shift test set, the results show an F1 score of 64.9% and an AUC of 80.3%. After excluding 30% of the high-uncertainty samples, these metrics improve to an F1 score of 74.4% and an AUC of 85.6%. CONCLUSION: Redirecting samples with high uncertainty to experts for subsequent diagnosis significantly decreases the rates of misdiagnosis, which highlights that uncertainty estimation is vital to ensure safe decision making for computer-aided early oral cancer diagnosis.

FSH induces EMT in ovarian cancer via ALKBH5-regulated Snail m6A demethylation.

Background: The therapeutic benefits of targeting follicle-stimulating hormone (FSH) receptor in treatment of ovarian cancer are significant, whereas the role of FSH in ovarian cancer progresses and the underlying mechanism remains to be developed. Methods: Tissue microarray of human ovarian cancer, tumor xenograft mouse model, and in vitro cell culture were used to investigate the role of FSH in ovarian carcinogenesis. siRNA, lentivirus and inhibitors were used to trigger the inactivation of genes, and plasmids were used to increase transcription of genes. Specifically, pathological characteristic was assessed by histology and immunohistochemistry (IHC), while signaling pathway was studied using western blot, quantitative RT-PCR, and immunofluorescence. Results: Histology and IHC of human normal ovarian and tumor tissue confirmed the association between FSH and Snail in ovarian cancer metastasis. Moreover, in epithelial ovarian cancer cells and xenograft mice, FSH was showed to promote epithelial mesenchymal transition (EMT) progress and metastasis of ovarian cancer via prolonging the half-life of Snail mRNA in a N6-methyladenine methylation (m6A) dependent manner, which was mechanistically through the CREB/ALKBH5 signaling pathway. Conclusions: These findings indicated that FSH induces EMT progression and ovarian cancer metastasis via CREB/ALKBH5/Snail pathway. Thus, this study provided new insight into the therapeutic strategy of ovarian cancer patients with high level of FSH.

Design, Synthesis and Anti-cancer Evaluation of Nitrogen-containing Derivatives of 30-Carboxyl of Gambogic Acid.

BACKGROUND: Gambogic acid (GA) is a natural product from the resin of the Garcinia species, which showed significant activity in the induction of apoptosis. .t can be one promising lead compound for the design and synthesis of new anticancer drugs. OBJECTIVE: The objective of the current study is to design novel nitrogen-contained GA derivatives with better anti-cancer activities and study the effect of the introduction of different nitrogen-contained groups on the activity of GA. METHODS: The designed 15 derivatives were synthesized via esterification or amidation of 30-carboxylate. The synthetic compounds were characterized via different spectroscopic techniques, including X-ray single crystal diffraction, MS and NMR. The cytotoxic activity of the designed derivatives was evaluated in vitro against A549, HepG-2, and MCF-7 cell lines using methyl thiazolyl tetrazolium (MTT) test. RESULTS: 15 nitrogen-contained GA derivatives were successfully synthesized and established. Based on the IC50 values, compounds 9, 10, 11 and 13 showed stronger inhibitory effects on A549, HepG-2, MCF-7 cell lines than GA, while 9 is the most active compound with IC50 value of 0.64-1.49 µM. Most derivatives of GA with esterification of C-30 including cyano-benzene ring were generally weaker than those of pyrimidinyl-substituted derivatives. In addition, length of alkyl linkers between C-30 of GA and nitrogen-contained group produced different effects on A549, HepG-2 and MCF-7 cell lines. CONCLUSION: The structure-activity relationship results show that aromatic substituent and linker length play important roles to improve the anticancer activities, while compound 9 with pyrimidine substituent and C-C-C linkers is the most active derivative against tested cell lines, and is a promising anti-cancer agent for further development.

Cathelicidin Antimicrobial Peptide Acts as a Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

Mutation of aspartic acid 262 on estrogen receptor abrogates estradiol signaling pathway.

OBJECTIVE: ERα (estrogen receptor alpha) exerts nuclear genomic actions and membrane-initiated non-genomic effects. The mutation of aspartic acid into alanine in vitro revealed the critical role of aspartic acid 258 (corresponding to mouse amino acid site 262) of ERα for non-nuclear function. Our previous in vitro study revealed that this mutation blocked estrogen's non-genomic effects on vascular endothelial H2S release. Here, we studied the in vivo role of the aspartic acid 262 of ERα in the reproductive system and in the vascular tissue. APPROACH AND RESULTS: We generated a mouse model harboring a point mutation of the murine counterpart of this aspartic acid into alanine (ERαD262A). Our results showed that the ERαD262A females are fertile with standard hormonal serum levels, but the uterine development and responded with estrogen and follicular development are disrupted. In line with our previous study, we found that the rapid dilation of the aorta was abrogated in ERαD262A mice. In contrast to the previously reported R264-ERα mice, the classical estrogen genomic effector SP1/NOS3/AP1 and the nongenomic effectors p-eNOs, p-AKT, and p-ERK were disturbed in the ERαD262A aorta. Besides, the serum H2S concentration was decreased in ERαD262A mice. Together, ERαD262A mice showed compromised both genomic and non-genomic actions in response to E2. CONCLUSIONS: These data showed that aspartic acid 262 of ERα are important for both genomic and non-genomic effects of E2. Our data provide a theoretical basis for further selecting an effective non-genomic mouse model and provide a new direction for developing estrogen non-genomic effect inhibitors.

Identification of key genes in the pathogenesis of preeclampsia via bioinformatic analysis and experimental verification.

Background: Preeclampsia (PE) is the primary cause of perinatal maternal-fetal mortality and morbidity. The exact molecular mechanisms of PE pathogenesis are largely unknown. This study aims to identify the hub genes in PE and explore their potential molecular regulatory network. Methods: We downloaded the GSE148241, GSE190971, GSE74341, and GSE114691 datasets for the placenta and performed a differential expression analysis to identify hub genes. We performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO), Gene Set Enrichment Analysis (GSEA), and Protein-Protein Interaction (PPI) Analysis to determine functional roles and regulatory networks of differentially expressed genes (DEGs). We then verified the DEGs at transcriptional and translational levels by analyzing the GSE44711 and GSE177049 datasets and our clinical samples, respectively. Results: We identified 60 DEGs in the discovery phase, consisting of 7 downregulated genes and 53 upregulated genes. We then identified seven hub genes using Cytoscape software. In the verification phase, 4 and 3 of the seven genes exhibited the same variation patterns at the transcriptional level in the GSE44711 and GSE177049 datasets, respectively. Validation of our clinical samples showed that CADM3 has the best discriminative performance for predicting PE. Conclusion: These findings may enhance the understanding of PE and provide new insight into identifying potential therapeutic targets for PE.

Exploring the Regulatory Role of XIST-microRNAs/mRNA Network in Circulating CD4+ T Cells of Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the main cause of cancer-related death globally. Immune dysregulation of CD4+ T cells has been identified to play a role in the development of HCC. Nevertheless, the underlying molecular pathways of CD4+ T cells in HCC are not completely known. Thus, a better understanding of the dysregulation of the lncRNA-miRNA/mRNA network may yield novel insights into the etiology or progression of HCC. In this study, circulating CD4+ T cells were isolated from the whole blood of 10 healthy controls and 10 HCC patients for the next-generation sequencing of the expression of lncRNAs, miRNAs, and mRNAs. Our data showed that there were different expressions of 34 transcripts (2 lncRNAs, XISTs, and MIR222HGs; 29 mRNAs; and 3 other types of RNA) and 13 miRNAs in the circulating CD4+ T cells of HCC patients. The expression of lncRNA-XIST-related miRNAs and their target mRNAs was confirmed using real-time quantitative polymerase chain reaction (qPCR) on samples from 100 healthy controls and 60 HCC patients. The lncRNA-miRNA/mRNA regulation network was created using interaction data generated from ENCORI and revealed there are positive correlations in the infiltration of total CD4+ T cells, particularly resting memory CD4+ T cells, and negative correlations in the infiltration of Th1 CD4+ T cells.

Deciphering the Divergent Gene Expression Landscapes of m6A/m5C/m1A Methylation Regulators in Hepatocellular Carcinoma Through Single-Cell and Bulk RNA Transcriptomic Analysis.

Introduction: RNA modifications mediated by the m6A, m1A, and m5C regulatory genes are crucial for the progression of malignancy. This study aimed to explore the expression of regulator genes for m6A/m5C/m1A methylation at the single-cell level and to validate their expression in cancerous and adjacent para-cancerous liver tissues of adult patients with HCC who underwent tumor resection. Methods: The bulk sequencing from The Cancer Genome Atlas (TCGA) database and the single-cell RNA sequencing (scRNA-seq) data obtained from the Gene Expression Omnibus (GEO) database were used to identify the dysregulated m6A/m5C/m1A genes for hepatocellular carcinoma (HCC). A real-time polymerase chain reaction (real-time PCR) was used to measure the expression of dysregulated m6A/m5C/m1A genes in collected human HCC tissues and compared with adjacent para-cancerous liver tissues. Immune cell infiltration with these significantly expressed methylation-related genes was evaluated using Timer2.0. Results: A discrepancy in m6A/m5C/m1A gene expression was observed between bulk sequencing and scRNA-seq. The clustered heatmap of the scRNA-seq-identified dysregulated m6A/m5C/m1A genes in TCGA cohort revealed heterogeneous expression of these methylation regulators within the cancer, whereas their expression in the adjacent liver tissues was more homogeneous. The real-time PCR validated the significant overexpression of DNMT1, NSUN5, TRMT6, IGF2BP1, and IGFBP3, which were identified using scRNA-seq, and IGFBP2, which was identified using bulk sequencing. These dysregulated methylation genes are mainly correlated with the infiltration of natural killer cells. Discussion: This study suggests that cellular diversity inside tumors contributes to the discrepancy in the expression of methylation regulator genes between traditional bulk sequencing and scRNA-seq. This study identified five regulatory genes that will be the focus of further studies regarding the function of m6A/m5C/m1A in HCC.

The System Profile of Renal Drug Transporters in Tubulointerstitial Fibrosis Model and Consequent Effect on Pharmacokinetics.

With the widespread clinical use of drug combinations, the incidence of drug-drug interactions (DDI) has significantly increased, accompanied by a variety of adverse reactions. Drug transporters play an important role in the development of DDI by affecting the elimination process of drugs in vivo, especially in the pathological state. Tubulointerstitial fibrosis (TIF) is an inevitable pathway in the progression of chronic kidney disease (CKD) to end-stage renal disease. Here, the dynamic expression changes of eleven drug transporters in TIF kidney have been systematically investigated. Among them, the mRNA expressions of Oat1, Oat2, Oct1, Oct2, Oatp4C1 and Mate1 were down-regulated, while Oat3, Mrp2, Mrp4, Mdr1-α, Bcrp were up-regulated. Pearson correlation analysis was used to analyze the correlation between transporters and Creatinine (Cr), OCT2 and MATE1 showed a strong negative correlation with Cr. In contrast, Mdr1-α exhibited a strong positive correlation with Cr. In addition, the pharmacokinetics of cimetidine, ganciclovir, and digoxin, which were the classical substrates for OCT2, MATE1 and P-glycoprotein (P-gp), respectively, have been studied. These results reveal that changes in serum creatinine can indicate changes in drug transporters in the kidney, and thus affect the pharmacokinetics of its substrates, providing useful information for clinical use.

Regulation of Macrophage Polarization by Mineralized Collagen Coating to Accelerate the Osteogenic Differentiation of Mesenchymal Stem Cells.

Osteogenesis on the interface between the implant and host bone is a synergistic processing of multiple systems involved in immune response, angiogenesis, osteogenesis, etc. However, regulation of the osteoimmune microenvironment on the implant surface to accelerate the osteogenesis through manipulating the polarization of macrophage phenotype is still beginning to be explored. We here demonstrate that macrophage phenotype is able to be regulated by decoration of mineralized collagen (MC) coating on the titanium implant surface via triggering the integrin-related cascade pathway of macrophages. Furthermore, regulation of the macrophage polarization and construction of the osteoimmune microenvironment by MC coating would subsequently accelerate the osteogenic differentiation of the mesenchymal stem cells. This work therefore emphasizes the importance of the osteoimmune microenvironment on osteogenesis and provides a promising strategy to improve the osteointegration of implants.

Exercise effects on fatigue in breast cancer survivors after treatments: A systematic review and meta-analysis.

AIM: The aim of this study is to explore the effects of exercise interventions by type, duration and intensity of exercise for fatigue in breast cancer survivors who had completed their treatment. BACKGROUND: Most studies found that exercise has valuable outcomes for cancer survivors. This meta-analysis comprehensively summarizes the benefits of exercise intervention for fatigue in breast cancer patients who had completed their adjuvant treatments. METHODS: We conducted a meta-analysis on randomized control trials published during 1 January 2000 through 31 December 2019, from PubMed, Cochrane Library databases, EMBASE, Medline (ProQuest), CINAHL, PsycINFO, Chinese Electronic Periodical Service and Wan Fan Data with prespecified searching criteria. Breast cancer patients earlier than stage IIIc and completing adjuvant treatments were included, and the effects of exercise on fatigue were investigated. RESULTS: Nine randomized controlled trials (RCTs) were included (N = 581). Patients receiving exercise interventions showed reduced fatigue comparing with those without exercise. Exercise with low-moderate intensity, 20 min/day, three times per week and lasting up to 12 weeks had a significant effect on reducing fatigue for breast cancer survivors. CONCLUSION: Our study suggested that exercise interventions can reduce fatigue for this group of cancer survivors. The duration and intensity of exercise intervention could be prescribed for this specific group of cancer patients as a basic requirement to handle their reported fatigue.

Loss of myeloid cell-specific SIRPα, but not CD47, attenuates inflammation and suppresses atherosclerosis.

AIMS: Inhibitors of the anti-phagocytic CD47-SIRPα immune checkpoint are currently in clinical development for a variety of haematological and solid tumours. Application of immune checkpoint inhibitors to the cardiovascular field is limited by the lack of preclinical studies using genetic models of CD47 and SIRPα inhibition. In this study, we comprehensively analysed the effects of global and cell-specific SIRPα and CD47 deletion on atherosclerosis development. METHODS AND RESULTS: Here, we show that both SIRPα and CD47 expression are increased in human atherosclerotic arteries and primarily co-localize to CD68+ areas in the plaque region. Hypercholesterolaemic mice homozygous for a Sirpa mutant lacking the signalling cytoplasmic region (Sirpamut/mut) and myeloid cell-specific Sirpa-knockout mice are protected from atherosclerosis. Further, global Cd47-/- mice are protected from atherosclerosis but myeloid cell-specific deletion of Cd47 increased atherosclerosis development. Using a combination of techniques, we show that loss of SIRPα signalling in macrophages stimulates efferocytosis, reduces cholesterol accumulation, promotes lipid efflux, and attenuates oxidized LDL-induced inflammation in vitro and induces M2 macrophage phenotype and inhibits necrotic core formation in the arterial wall in vivo. Conversely, loss of myeloid cell CD47 inhibited efferocytosis, impaired cholesterol efflux, augmented cellular inflammation, stimulated M1 polarization, and failed to decrease necrotic core area in atherosclerotic vessels. Finally, comprehensive blood cell analysis demonstrated lower haemoglobin and erythrocyte levels in Cd47-/- mice compared with wild-type and Sirpamut/mut mice. CONCLUSION: Taken together, these findings identify SIRPα as a potential target in atherosclerosis and suggest the importance of cell-specific CD47 inhibition as a future therapeutic strategy.

MEK inhibition exerts temporal and myeloid cell-specific effects in the pathogenesis of neurofibromatosis type 1 arteriopathy.

Mutations in the NF1 tumor suppressor gene are linked to arteriopathy. Nf1 heterozygosity (Nf1+/-) results in robust neointima formation, similar to humans, and myeloid-restricted Nf1+/- recapitulates this phenotype via MEK-ERK activation. Here we define the contribution of myeloid subpopulations to NF1 arteriopathy. Neutrophils from WT and Nf1+/- mice were functionally assessed in the presence of MEK and farnesylation inhibitors in vitro and neutrophil recruitment to lipopolysaccharide was assessed in WT and Nf1+/- mice. Littermate 12-15 week-old male wildtype and Nf1+/- mice were subjected to carotid artery ligation and provided either a neutrophil depleting antibody (1A8), liposomal clodronate to deplete monocytes/macrophages, or PD0325901 and neointima size was assessed 28 days after injury. Bone marrow transplant experiments assessed monocyte/macrophage mobilization during neointima formation. Nf1+/- neutrophils exhibit enhanced proliferation, migration, and adhesion via p21Ras activation of MEK in vitro and in vivo. Neutrophil depletion suppresses circulating Ly6Clow monocytes and enhances neointima size, while monocyte/macrophage depletion and deletion of CCR2 in bone marrow cells abolish neointima formation in Nf1+/- mice. Taken together, these findings suggest that neurofibromin-MEK-ERK activation in circulating neutrophils and monocytes during arterial remodeling is nuanced and points to important cross-talk between these populations in the pathogenesis of NF1 arteriopathy.

Laser-Assisted Drug Delivery of Tranexamic Acid by Picosecond Laser in Postinflammatory Hyperpigmentation: A Split-Area Double Blind Randomized Prospective Study.

Background: Tranexamic acid has been reported to benefit the treatment of postinflammatory hyperpigmentation (PIH). Laser-assisted drug delivery (LADD) could facilitate the efficacy of topically applied drugs into the dermis. This split-area randomized prospective study aims to assess whether early utilization of the LADD procedure with tranexamic acid delivery followed by picosecond lasers can attenuate the PIH better than the utilization of picosecond lasers alone. Patients and methods: Ten post-traumatic cases of PIH in 10 patients (8 female and 2 male) with an average age of 34.2 ± 11.2 years were included in this clinical trial. Using block randomization to determine the treatment side, one side of each area of the PIH was separated from the midline into two halves belonging to the control and tranexamic acid groups. The half area of the tranexamic acid group was further topically applied with 10% tranexamic acid solution. This procedure was repeated every 6 weeks, four times in total. Results: The self-assessment of the hyperpigmentation and overall satisfaction of the treatment outcome were not significantly different between the treatment and control sides. Conclusions: This split-area study revealed that, compared with picosecond alone, there was no significant difference adopting tranexamic acid in LADD after nonablative fractional picosecond laser for PIH.

Automatic detection of oral cancer in smartphone-based images using deep learning for early diagnosis.

SIGNIFICANCE: Oral cancer is a quite common global health issue. Early diagnosis of cancerous and potentially malignant disorders in the oral cavity would significantly increase the survival rate of oral cancer. Previously reported smartphone-based images detection methods for oral cancer mainly focus on demonstrating the effectiveness of their methodology, yet it still lacks systematic study on how to improve the diagnosis accuracy on oral disease using hand-held smartphone photographic images. AIM: We present an effective smartphone-based imaging diagnosis method, powered by a deep learning algorithm, to address the challenges of automatic detection of oral diseases. APPROACH: We conducted a retrospective study. First, a simple yet effective centered rule image-capturing approach was proposed for collecting oral cavity images. Then, based on this method, a medium-sized oral dataset with five categories of diseases was created, and a resampling method was presented to alleviate the effect of image variability from hand-held smartphone cameras. Finally, a recent deep learning network (HRNet) was introduced to evaluate the performance of our method for oral cancer detection. RESULTS: The performance of the proposed method achieved a sensitivity of 83.0%, specificity of 96.6%, precision of 84.3%, and F1 of 83.6% on 455 test images. The proposed "center positioning" method was about 8% higher than that of a simulated "random positioning" method in terms of F1 score, the resampling method had additional 6% of performance improvement, and the introduced HRNet achieved slightly better performance than VGG16, ResNet50, and DenseNet169, with respect to the metrics of sensitivity, specificity, precision, and F1. CONCLUSIONS: Capturing oral images centered on the lesion, resampling the cases in training set, and using the HRNet can effectively improve the performance of deep learning algorithm on oral cancer detection. The smartphone-based imaging with deep learning method has good potential for primary oral cancer diagnosis.

Investigate the improvement of facial skin texture with the VISIA system after total thyroidectomy.

BACKGROUND: During clinical practice we have noticed that some patients with hyperthyroidism have finer skin with less wrinkles, pores, and spots after thyroidectomy, and the improvement can be observed within a few weeks after the operation. However, there is no evidence or study in the literature to proof this finding. AIM AND OBJECTIVE: This study was designed to evaluate and quantify the skin characters of patients with hyperthyroidism before and after thyroidectomy. MATERIAL AND METHODS: This is a prospective study to include patients with hyperthyroidism who received total thyroidectomy between March 1st, 2018 and February 28th, 2019. The patients received blood test for T4 and TSH analysis and VISIA measurements for skin texture quantification, at the preoperative stage, three, and six months postoperatively. A total of 8 patients were included. Repeated measurement was used to determine the lab data and VISIA measurement changes before and after the operation. Mauchly's sphericity test was performed to determine whether the violation of sphericity occurs, and the Greenhouse-Geisser correction was used when the violation of sphericity occurs. RESULTS: All the patients were female and generally healthy without systemic medical disease except the hyperthyroidism. The T4 and TSH levels were not significantly different before and after the thyroidectomy. In terms of the skin character measurements, the wrinkles, texture, pores, UV spots, and brown spots were not improved after thyroidectomy. A trend of improvement in spots, red area, and porphyrin was noted, although not statistically significant. CONCLUSIONS: Surgical removal of the thyroid gland in patients with hyperthyroidism does not improve the skin quality and texture in examinations via the VISIA system.

Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis.

AIMS: Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes (LNs) and development of atherosclerosis. METHODS AND RESULTS: The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells (LEC) in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signalling in LEC. In vivo low-density lipoprotein (LDL) tracking and perivascular blockade of RSPO2-LGR4 signalling using LGR4-extracellular domain (ECD) pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2-LGR4 signalling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 suppresses PI3K-AKT-endothelial nitric oxide synthase (eNOS) signalling via LGR4 and inhibits activation of the canonical Wnt-ß-catenin pathway. ApoE-/- mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently labelled LDL to deep cervical LNs were observed in LGR4-ECD-treated mice. CONCLUSION: These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-nitric oxide signalling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries.

Caffeic acid phenethyl ester suppresses androgen receptor signaling and stability via inhibition of phosphorylation on Ser81 and Ser213.

BACKGROUND: Androgen receptor (AR) plays important role in the development, progression, and metastasis of prostate cancer (PCa). Caffeic acid phenethyl ester (CAPE) is the main component of honey bee propolis. We determined if CAPE affects the signaling and stability of AR in PCa cells. METHODS: Effects of CAPE on AR transcriptional activity and localization were determined by reporter gene assay and immunofluorescent microscopy. Western blotting, fluorescent polarization, computer simulation, and animal experiment were performed to investigate the molecular mechanism how CAPE reduces the stability of AR. RESULTS: CAPE treatment dose-dependently suppressed the transcriptional activity of AR as well as the protein levels of AR and its target gene PSA. Cyclohexamide treatment revealed that androgen stabilized AR protein, but AR stability was diminished by CAPE. Fluorescence microscopy demonstrated that androgen promoted the nucleus translocation of AR in PCa cells, while treatment with CAPE reduced protein level of AR in both nucleus and cytoplasm. CAPE treatment suppressed the phosphorylation of Ser81 and Ser213 on AR, which regulates the stability of AR. CDK1 and AKT are the kinases phosphorylating Ser81 and Ser213 on AR, respectively. CAPE treatment significantly reduced the protein level and activity of CDK1 and AKT in PCa cells. Overexpression of CDK1 or AKT rescued the AR protein level under CAPE treatment. CONCLUSIONS: Our results suggested that CAPE treatment reduced AR stability and AR transcriptional activity in PCa cells, implying the possibility of using CAPE as a treatment for advanced PCa.

Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis.

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/-) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/- mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/- or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.

PKCδ stimulates macropinocytosis via activation of SSH1-cofilin pathway.

Macropinocytosis is an actin-dependent endocytic mechanism mediating internalization of extracellular fluid and associated solutes into cells. The present study was designed to identify the specific protein kinase C (PKC) isoform(s) and downstream effectors regulating actin dynamics during macropinocytosis. We utilized various cellular and molecular biology techniques, pharmacological inhibitors and genetically modified mice to study the signaling mechanisms mediating macropinocytosis in macrophages. The qRT-PCR experiments identified PKCδ as the predominant PKC isoform in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated the functional role of PKCδ in phorbol ester- and hepatocyte growth factor (HGF)-induced macropinocytosis. Western blot analysis demonstrated that phorbol ester and HGF stimulate activation of slingshot phosphatase homolog 1 (SSH1) and induce cofilin Ser-3 dephosphorylation via PKCδ in macrophages. Silencing of SSH1 inhibited cofilin dephosphorylation and macropinocytosis stimulation. Interestingly, we also found that incubation of macrophages with BMS-5, a potent inhibitor of LIM kinase, does not stimulate macropinocytosis. In conclusion, the findings of the present study demonstrate a previously unidentified mechanism by which PKCδ via activation of SSH1 and cofilin dephosphorylation stimulates membrane ruffle formation and macropinocytosis. The results of the present study may contribute to a better understanding of the regulatory mechanisms during macrophage macropinocytosis.