SNHG18 controls vascular smooth muscle cell contractile phenotype and neointimal hyperplasia.

AIMS: Long non-coding RNA (LncRNA) small nucleolar RNA host gene 18 (SNHG18) has been widely implicated in cancers. However, little is known about its functional involvement in vascular diseases. Herein, we attempted to explore a role for SNHG18 in modulating vascular smooth muscle cell (VSMC) contractile phenotype and injury-induced neointima formation. METHODS AND RESULTS: Analysis of single cell RNA sequencing and transcriptomic datasets showed decreased levels of SNHG18 in injured and atherosclerotic murine and human arteries, which is positively associated with VSMC contractile genes. SNHG18 was upregulated in VSMCs by TGFß1 through transcription factors Sp1 and SMAD3. SNHG18 gene gain/loss-of-function studies revealed that VSMC contractile phenotype was positively regulated by SNHG18. Mechanistic studies showed that SNHG18 promotes a contractile VSMC phenotype by up-regulating miR-22-3p. SNHG18 up-regulates miR-22 biogenesis and miR-22-3p production by competitive binding with the A-to-I RNA editing enzyme, adenosine deaminase acting on RNA-2 (ADAR2). Surprisingly, we observed that ADAR2 inhibited miR-22 biogenesis not through increasing A-to-I editing within primary miR-22, but by interfering the binding of microprocessor complex subunit DGCR8 to primary miR-22. Importantly, perivascular SNHG18 overexpression in the injured vessels dramatically up-regulated the expression levels of miR-22-3p and VSMC contractile genes, and prevented injury-induced neointimal hyperplasia. Such modulatory effects were reverted by miR-22-3p inhibition in the injured arteries. Finally, we observed a similar regulator role for SNHG18 in human VSMCs, and a decreased expression level of both SNHG18 and miR-22-3p in diseased human arteries; and we found that the expression level of SNHG18 was positively associated with that of miR-22-3p in both healthy and diseased human arteries. CONCLUSION: We demonstrate that SNHG18 is a novel regulator in governing VSMC contractile phenotype and preventing injury-induced neointimal hyperplasia. Our findings have important implications for therapeutic targeting snhg18/miR-22-3p signalling in vascular diseases.

Tree shrews as a new animal model for systemic sclerosis research.

Objective: Systemic sclerosis (SSc) is a chronic systemic disease characterized by immune dysregulation and fibrosis for which there is no effective treatment. Animal models are crucial for advancing SSc research. Tree shrews are genetically, anatomically, and immunologically closer to humans than rodents. Thus, the tree shrew model provides a unique opportunity for translational research in SSc. Methods: In this study, a SSc tree shrew model was constructed by subcutaneous injection of different doses of bleomycin (BLM) for 21 days. We assessed the degree of inflammation and fibrosis in the skin and internal organs, and antibodies in serum. Furthermore, RNA sequencing and a series of bioinformatics analyses were performed to analyze the transcriptome changes, hub genes and immune infiltration in the skin tissues of BLM induced SSc tree shrew models. Multiple sequence alignment was utilized to analyze the conservation of selected target genes across multiple species. Results: Subcutaneous injection of BLM successfully induced a SSc model in tree shrew. This model exhibited inflammation and fibrosis in skin and lung, and some developed esophageal fibrosis and secrum autoantibodies including antinuclear antibodies and anti-scleroderma-70 antibody. Using RNA sequencing, we compiled skin transcriptome profiles in SSc tree shrew models. 90 differentially expressed genes (DEGs) were identified, which were mainly enriched in the PPAR signaling pathway, tyrosine metabolic pathway, p53 signaling pathway, ECM receptor interaction and glutathione metabolism, all of which are closely associated with SSc. Immune infiltration analysis identified 20 different types of immune cells infiltrating the skin of the BLM-induced SSc tree shrew models and correlations between those immune cells. By constructing a protein-protein interaction (PPI) network, we identified 10 hub genes that were significantly highly expressed in the skin of the SSc models compared to controls. Furthermore, these genes were confirmed to be highly conserved in tree shrews, humans and mice. Conclusion: This study for the first time comfirmed that tree shrew model of SSc can be used as a novel and promising experimental animal model to study the pathogenesis and translational research in SSc.

Associations between marital quality and the prognosis of breast cancer in young Chinese women: 10.3-year median follow-up.

BACKGROUND: Some evidence has revealed that marital status is an important predictor of breast cancer (BC) prognosis. However, what role marital quality plays in the effect of marital status on BC prognosis remains unclear. METHODS: We conducted a prospective cohort study of women aged 20-50 years with stage I-III BC treated in accordance with a standard treatment protocol. The following three categories of marital quality were assessed: marital satisfaction, sexual relationship, and couple communication. The log-rank test was used to compare survival. Cox proportional hazards models were used to estimate hazard ratio (HR) and 95% confidence interval (CI) for recurrence and metastasis, BC-specific mortality, and overall mortality, adjusting for clinical variables. RESULTS: A total of 1,043 married women were initially recruited in the study. Forty-five (4.3%) patients refused to participate in this study and 141 (13.5%) were excluded from the analysis. Among 857 participants, there were 59 deaths, including 57 from BC. Multivariate Cox regression analysis showed that patients with poor marital satisfaction had significantly higher risks of recurrence and metastasis (HR 3.942, 95% CI: 1.903-8.167), BC-specific mortality (HR 3.931, 95% CI: 1.896-8.150), and overall mortality (HR 3.916, 95% CI: 1.936-7.924). Those with poor sexual relationship had significantly higher risks of recurrence and metastasis (HR 5.763, 95% CI: 3.012-11.027), BC-specific mortality (HR 5.724, 95% CI: 2.992-10.949), and overall mortality (HR 5.653, 95% CI: 2.993-10.680). CONCLUSIONS: Our results identified a subset of BC patients who have a poor prognosis, namely, those with poor marital quality. Early screening for marital quality and applying necessary social support interventions are helpful in improving the prognosis of patients with poor marital quality.

A meta-analysis of the clinical significance of neutrophil-to-lymphocyte ratios in interstitial lung disease.

Interstitial lung disease (ILD) is a group of diffuse parenchymal infiltrating diseases of different etiologies. The neutrophil-to-lymphocyte ratio (NLR) can reflect ILD's existence, progression, and prognosis and is currently regarded as a promising biological marker. This meta-analysis assessed elevated NLR levels in ILD for their predictive value. From inception to July 27, 2022, the Scopus, Cochrane Library, Web of Science, Embase, and PubMed databases were checked thoroughly. We used the weighted mean difference (WMD) and 95% confidence interval (CI) to compare blood NLR values between groups. We examined the relationship between poor prognoses and elevated NLR concentrations in ILD patients using odds ratios (ORs) and 95% CI. After initially including 443 studies, 24 were ultimately analyzed. Fifteen studies(ILD:n = 2,912, Non-ILD: n = 2,868) revealed that the NLR values in the ILD group were relatively high (WMD = 0.61, 95% CI 0.43-0.79, p = 0.001). Eight articles (with poor prognoses: n = 407, without poor prognoses: n = 340) indicated that ILD patients with poor prognoses had higher NLR values (WMD = 1.33, 95% CI 0.32-2.33, p = 0.01). This distinction was especially noticeable in patients with the connective tissue disease (CTD)associated with ILD subgroup (WMD = 3.53, 95% CI 1.54-5.51, p = 0.0005). The pooled OR for increased NLR levels forecasting poor prognoses of ILD was 1.09 (95% CI 1.03-1.15, p = 0.0008). Increasing blood NLR values have clinical significance and application value for detecting ILD and predicting its poor prognosis, especially in CTD patients.

Structural Characterization of Thiadiazolesulfonamide Inhibitors Bound to Neisseria gonorrhoeae α-Carbonic Anhydrase.

Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.

Structure-activity relationship studies for inhibitors for vancomycin-resistant Enterococcus and human carbonic anhydrases.

Vancomycin-resistant enterococci (VRE), consisting of pathogenic Enterococcus faecalis and E. faecium, is a leading cause of hospital-acquired infections (HAIs). We recently repurposed the FDA-approved human carbonic anhydrase (CA) inhibitor acetazolamide (AZM) against VRE agent with the likely mechanism of action for the molecules being inhibition of one, or both, of the bacterial CA isoforms expressed in VRE. To elucidate how inhibitor binding to the enzymes relates to MIC, we further characterised the inhibition constants (Ki) against the E. faecium α-CA (Efα-CA) and γ-CA (Efγ-CA), as well as against human CA I (hCAI) and human CA II (hCAII) to assess selectivity. We have also utilised homology modelling and molecular dynamics (MD) simulations to gain a better understanding of the potential interactions the molecules are making with the targets. In this paper, we elaborate on the SAR for the AZM analogs as it pertains to MIC and Ki for each CA.

The role of splicing factor PRPF8 in breast cancer.

BACKGROUND: Alternative splicing is a mechanism to produce different proteins with diverse functions from one gene. Many splicing factors play an important role in cancer progression. PRPF8 is a core protein component of the spliceosome complex, U4/U6-U5 tri-snRNP. OBJECTIVE: However, PRPF8 involved in mRNA alternative splicing are rarely included in the prognosis. METHODS: We found that PRPF8 was expressed in all examined cancer types. Further analyses found that PRPF8 expression was significantly different between the breast cancer and paracancerous tissues. RESULTS: Survival analyses showed that PRPF8-high patients had a poor prognosis, and the expression of PRPF8 is associated with distant metastasis-free survival (DMFS) and post progression survival (PPS). Gene Set Enrichment Analysis (GSEA) has revealed that PRPF8 expression is correlated with TGF-ß, JAK-STAT, and cell cycle control pathways. Consistent with these results, upon PRPF8 silencing, the growth of MCF-7 cells was reduced, the ability of cell clone formation was weakened, and p⁢21 expression was increased. CONCLUSIONS: These results have revealed that PRPF8 is a significant factor for splicing in breast cancer progression.

Cezanne is a critical regulator of pathological arterial remodelling by targeting ß-catenin signalling.

AIMS: Pathological arterial remodelling including neointimal hyperplasia and atherosclerosis is the main underlying cause for occluding arterial diseases. Cezanne is a novel deubiquitinating enzyme, functioning as a NF-кB negative regulator, and plays a key role in renal inflammatory response and kidney injury induced by ischaemia. Here we attempted to examine its pathological role in vascular smooth muscle cell (VSMC) pathology and arterial remodelling. METHODS AND RESULTS: Cezanne expression levels were consistently induced by various atherogenic stimuli in VSMCs, and in remodelled arteries upon injury. Functionally, VSMCs over-expressing wild-type Cezanne, but not the mutated catalytically-inactive Cezanne (C209S), had an increased proliferative ability and mobility, while the opposite was observed in VSMCs with Cezanne knockdown. Surprisingly, we observed no significant effects of Cezanne on VSMC apoptosis, NF-κB signalling, or inflammation. RNA-sequencing and biochemical studies showed that Cezanne drives VSMC proliferation by regulating CCN family member 1 (CCN1) by targeting ß-catenin for deubiquitination. Importantly, local correction of Cezanne expression in the injured arteries greatly decreased VSMC proliferation, and prevented arterial inward remodelling. Interestingly, global Cezanne gene deletion in mice led to smaller atherosclerotic plaques, but with a lower level of plaque stability. Translating, we observed a similar role for Cezanne in human VSMCs, and higher expression levels of Cezanne in human atherosclerotic lesions. CONCLUSION: Cezanne is a key regulator of VSMC proliferation and migration in pathological arterial remodelling. Our findings have important implications for therapeutic targeting Cezanne signalling and VSMC pathology in vascular diseases.

Neutrophil elastase promotes neointimal hyperplasia by targeting toll-like receptor 4 (TLR4)-NF-κB signalling.

BACKGROUND AND PURPOSE: Neointimal hyperplasia (NIH) is the fundamental cause for vascular diseases and vascular smooth muscle cell (VSMC) dysregulation has been widely implicated in NIH. Neutrophil elastase is a potential therapeutic target for multiple diseases. We investigated the role of neutrophil elastase in VSMC functions and injury-induced NIH and explored the therapeutic potential of targeting neutrophil elastase in NIH. EXPERIMENTAL APPROACH: VSMCs were used to analyse the effects of neutrophil elastase. Proteomic analysis was used to identify potential neutrophil elastase targets. Artery injury model and neutrophil elastase inhibitor GW311616A were used to investigate the role of neutrophil elastase in NIH. KEY RESULTS: TNF-α up-regulated neutrophil elastase in VSMCs through modulating GAPBα/Runx1/CEBPα/c-Myb signalling. Up-regulated neutrophil elastase promoted VSMC migration, proliferation and inflammation. Toll-like receptor 4 (TLR4) was identified as a target protein for neutrophil elastase in VSMCs and the TLR4/MyD88/IRAK1/TRAF6/NF-κB regulatory axis was shown to be the signalling pathway for neutrophil elastase in VSMC pathology. Importantly, TLR4 inhibition abolished neutrophil elastase-mediated VSMC dysregulation. Injury-induced NIH was significantly reduced in both neutrophil elastase-deficient mice and mice treated with GW311616A. The formation of neutrophil extracellular traps was impaired in injured arteries from neutrophil elastase-deficient mice. Finally, a similar role for neutrophil elastase in human VSMC pathology was confirmed and we observed higher expression levels of neutrophil elastase but lower expression levels of TLR4 in human atherosclerotic lesions. CONCLUSION AND IMPLICATIONS: We provide new insight into the molecular mechanisms underlying NIH and identify neutrophil elastase as a potential therapeutic target for vascular disease.

LRH1 Acts as an Oncogenic Driver in Human Osteosarcoma and Pan-Cancer.

Osteosarcoma (OS) that mainly occurs during childhood and adolescence is a devastating disease with poor prognosis presented by extreme metastases. Recent studies have revealed that liver receptor homolog 1 (LRH-1) plays a vital role in the metastasis of several human cancers, but its role is unknown in the metastasis of OS. In this study, Gene Ontology (GO) enrichment analyses based on high-throughput RNA-seq data revealed that LRH-1 acted a pivotal part in the positive regulation of cell migration, motility, and angiogenesis. Consistently, LRH-1 knockdown inhibited the migration of human OS cells, which was concurrent with the downregulation of mesenchymal markers and the upregulation of epithelial markers. In addition, short hairpin RNAs (shRNAs) targeting LRH-1 inactivated transforming growth factor beta (TGF-ß) signaling pathway. LRH-1 knockdown inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation. Vascular endothelial growth factor A (VEGFA) expression was also downregulated after LRH-1 knockdown. Immunohistochemistry (IHC) revealed that the expression of LRH-1 protein was significantly higher in tumor tissues than in normal bone tissues. We found that high LRH-1 expression was associated with poor differentiation and advanced TNM stage in OS patients using IHC. Based on The Cancer Genome Atlas (TCGA) database, high LRH-1 expression predicts poor survival in lung squamous cell carcinoma (LUSC), kidney renal papillary cell carcinoma (KIRP), and pancreatic adenocarcinoma (PAAD). The downregulation of LRH-1 significantly hindered the migration and motility of LUSC cells. Using multi-omic bioinformatics, the positive correlation between LRH-1- and EMT-related genes was found across these three cancer types. GO analysis indicated that LRH-1 played a vital role in "blood vessel morphogenesis" or "vasculogenesis" in KIRP. Our results indicated that LRH-1 plays a tumor-promoting role in human OS, could predict the early metastatic potential, and may serve as a potential target for cancer therapy.

Structure-Activity Relationship Studies of Acetazolamide-Based Carbonic Anhydrase Inhibitors with Activity against Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an urgent threat to public health in the United States and around the world. Many of the current classes of antibiotics to treat N. gonorrhoeae infection are quickly becoming obsolete due to increased rates of resistance. Thus, there is a critical need for alternative antimicrobial targets and new chemical entities. Our team has repurposed the FDA-approved carbonic anhydrase inhibitor scaffold of acetazolamide to target N. gonorrhoeae and the bacteria's essential carbonic anhydrase, NgCA. This study established both structure-activity and structure-property relationships that contribute to both antimicrobial activity and NgCA activity. This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 µg/mL equating to an 8- to 16-fold improvement in antigonococcal activity compared to acetazolamide. These analogues were determined to be bacteriostatic against the pathogen and likely on-target against NgCA. Additionally, they did not exhibit any detrimental effects in cellular toxicity assays against both a human endocervical (End1/E6E7) cell line or colorectal adenocarcinoma cell line (Caco-2) at concentrations up to 128 µg/mL. Taken together, this study presents a class of antigonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models.

Upregulation of microRNA-155 Enhanced Migration and Function of Dendritic Cells in Three-dimensional Breast Cancer Microenvironment.

Background: Dendritic cells (DCs) play an essential role in the induction and regulation of immune responses, including the activation of effector T lymphocytes for the eradication of cancers. However, the tumor microenvironment (TME) often leads to DCs dysfunction due to their immature state. MicroRNA-155 (miR-155) has emerged as a typical multifunctional gene regulator associated with immune system development and immune cell activation and differentiation.Methods: In this study, a three-dimensional TME model that closely mimics the microenvironment of breast cancer was prepared. MiR-155 overexpression and control vectors were constructed using lentivirus. The relative expression of miR-155 was determined by qRT-PCR. Cell viability, antigen uptake and cell surface marker expression were analyzed by live-dead staining and flow cytometry. The migration ability of bone marrow-derived DCs (BMDCs) was qualified by transwell assay. A mixed lymphocyte culture assay was used to assess T cell-specific proliferation. Cytokine levels were determined by ELISA.Results: We found that the expression of miR-155 in DCs was inhibited by the TME. Furthermore, upregulation of miR-155 enhanced the migration ability, uptake of antigen and elevated the expression of the mature DCs markers CD80 and MHCII. More importantly, overexpression of miR-155 in DCs significantly induced T cell proliferation and IFN-γ and IL-2 secretion.Conclusion: MiR-155 is a potential molecular regulator that may improve the efficacy of DCs-based tumor immunotherapy.

miRNA-200c-3p promotes endothelial to mesenchymal transition and neointimal hyperplasia in artery bypass grafts.

Increasing evidence has suggested a critical role for endothelial-to-mesenchymal transition (EndoMT) in a variety of pathological conditions. MicroRNA-200c-3p (miR-200c-3p) has been implicated in epithelial-to-mesenchymal transition. However, the functional role of miR-200c-3p in EndoMT and neointimal hyperplasia in artery bypass grafts remains largely unknown. Here we demonstrated a critical role for miR-200c-3p in EndoMT. Proteomics and luciferase activity assays revealed that fermitin family member 2 (FERM2) is the functional target of miR-200c-3p during EndoMT. FERMT2 gene inactivation recapitulates the effect of miR-200c-3p overexpression on EndoMT, and the inhibitory effect of miR-200c-3p inhibition on EndoMT was reversed by FERMT2 knockdown. Further mechanistic studies revealed that FERM2 suppresses smooth muscle gene expression by preventing serum response factor nuclear translocation and preventing endothelial mRNA decay by interacting with Y-box binding protein 1. In a model of aortic grafting using endothelial lineage tracing, we observed that miR-200c-3p expression was dramatically up-regulated, and that EndoMT contributed to neointimal hyperplasia in grafted arteries. MiR-200c-3p inhibition in grafted arteries significantly up-regulated FERM2 gene expression, thereby preventing EndoMT and reducing neointimal formation. Importantly, we found a high level of EndoMT in human femoral arteries with atherosclerotic lesions, and that miR-200c-3p expression was significantly increased, while FERMT2 expression levels were dramatically decreased in diseased human arteries. Collectively, we have documented an unexpected role for miR-200c-3p in EndoMT and neointimal hyperplasia in grafted arteries. Our findings offer a novel therapeutic opportunity for treating vascular diseases by specifically targeting the miR-200c-3p/FERM2 regulatory axis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Identification of spliceosome components pivotal to breast cancer survival.

Cancer cells employ alternative splicing (AS) to acquire splicing isoforms favouring their survival. However, the causes of aberrant AS in breast cancer are poorly understood. In this study, the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) data were analysed with univariate feature selection. Of 122 analysed spliceosome components, U2SURP, PUF60, DDX41, HNRNPAB, EIF4A3, and PPIL3 were significantly associated with breast cancer survival. The top 4 four genes, U2SURP, PUF60, DDX41, and HNRNPAB, were chosen for further analyses. Their expression was significantly associated with cancer molecular subtype, tumour stage, tumour grade, overall survival (OS), and cancer-specific survival in the METABRIC data. These results were verifiable using other cohorts. The Cancer Genome Atlas data unveiled the elevated expression of PUF60, DDX41, and HNRNPAB in tumours compared with the normal tissue and confirmed the differential expression of the four genes among cancer molecular subtypes, as well as the associations of U2SURP, PUF60, and DDX41 expression with tumour stage. A meta-analysis data verified the associations of U2SURP, PUF60, and HNRNPAB expression with tumour grade, the associations of PUF60, DDX41, and HNRNPAB expression with OS and distant metastasis-free survival, and the associations of U2SURP and HNRNPAB expression with relapse-free survival. Experimentally, we demonstrated that inhibiting the expression of the four genes separately suppressed cell colony formation and slowed down cell growth considerably in breast cancer cells, but not in immortal breast epithelial cells. In conclusion, we have identified U2SURP, PUF60, DDX41, and HNRNPAB are spliceosome-related genes pivotal for breast cancer survival.

The exosomes derived from CAR-T cell efficiently target mesothelin and reduce triple-negative breast cancer growth.

Genetically engineered T cells expressing a chimeric antigen receptor (CAR) have rapidly developed into a powerful and innovative therapeutic modality for cancer patients. However, the problem of dose-dependent systemic toxicity cannot be ignored. In this study, exosomes derived from mesothelin (MSLN)-targeted CAR-T cells were isolated, and we found that they maintain most characteristics of the parental T cells, including surface expression of the CARs and CD3. Furthermore, CAR-carrying exosomes significantly inhibited the growth of both endogenous and exogenous MSLN-positive triple-negative breast cancer (TNBC) cells. The expression of the effector molecules perforin and granzyme B may be a mechanism of tumor killing. More importantly, a highly effective tumor inhibition rate without obvious side effects was observed with the administration of CAR-T cell exosomes in vivo. Thus, the use of CAR-T cell exosomes has great therapeutic potential against MSLN-expressing TNBC.

miR-214-3p-Sufu-GLI1 is a novel regulatory axis controlling inflammatory smooth muscle cell differentiation from stem cells and neointimal hyperplasia.

BACKGROUND: Inflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia. METHODS: The role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries. RESULTS: miR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries. CONCLUSIONS: We present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.

Optimization of Acetazolamide-Based Scaffold as Potent Inhibitors of Vancomycin-Resistant Enterococcus.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 µg/mL (acetazolamide) to MIC = 0.007 µg/mL (22) and 1 µg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Macrophage-derived MMP-8 determines smooth muscle cell differentiation from adventitia stem/progenitor cells and promotes neointima hyperplasia.

AIMS: Emerging evidence has suggested that adventitia stem/progenitor cells (AdSPCs) migrate into the intima of arteries in response to injury, where they differentiate towards smooth muscle cells (SMCs) and participate in neointimal hyperplasia. We have previously identified matrix metalloproteinase-8 (MMP8) as a key player in atherogenesis. In this study, we aimed to investigate the functional roles of macrophage-derived MMP8 in AdSPC differentiation and injury-induced arterial remodelling. METHODS AND RESULTS: We first observed an important role for MMP8 in SMC differentiation from embryonic stem cells, but this effect was not seen in AdSPCs. Instead, through macrophages/AdSPCs co-culture and macrophage conditional culture medium studies, we have demonstrated that the MMP8 protein secreted from macrophages promotes SMC differentiation from AdSPCs. Mechanistically, we showed that macrophage-derived MMP8 promotes SMC differentiation from AdSPCs through modulating transforming growth factor-ß activity and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)/Notch1 signalling. We further demonstrated that the binding site for CBF1, Suppressor of Hairless, and Lag-1 (CSL) within SMC gene promoters is responsible for Notch1 mediated SMC differentiation. Finally, we demonstrated that macrophage-derived MMP8 increased injury-induced neointimal SMC hyperplasia by activating ADAM10/Notch1 signalling. CONCLUSIONS: We have identified macrophage-derived MMP8 as a regulator in SMC differentiation from AdSPCs and neointimal SMC hyperplasia in response to injury. Our data provide new insights into the roles of MMP8 in AdSPC differentiation and the pathogenesis of neointima formation in the context of angiographic restenosis, and therefore may aid in the development of novel therapeutic agents for the prevention of this disease.

Development of a Split Esterase for Protein-Protein Interaction-Dependent Small-Molecule Activation.

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small-molecule-based imaging agents are widely used in biological studies, and small-molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small-molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show that the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate that the esterase-based reporter system is compatible with other commonly used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small-molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show that the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

High Expression of Plakoglobin Promotes Metastasis in Invasive Micropapillary Carcinoma of the Breast via Tumor Cluster Formation.

Invasive micropapillary carcinoma of the breast (IMPC) is a rare subtype of breast cancer that has a high frequency of lymph node (LN) involvement and metastasis to distant organs. IMPC is characterized by distinct histomorphology and unfavorable prognosis when compared with invasive ductal carcinoma no special type (IDC-NST). However, the underlying molecular mechanisms remain unclear. We reported here that plakoglobin, as a key component in cell adhesion, can promote collective metastasis through facilitating IMPC clusters formation. In comparing the clinicopathological features of 451 IMPC patients and 282 IDC-NST patients, our results showed that tumor emboli were significantly higher in IMPC patients and were associated with a high frequency of metastasis. Both in vitro and in vivo data showed overexpression of plakoglobin in both the cell membrane and the cytoplasm of IMPC clusters. When plakoglobin was knocked down in IMPC cell models, the tumor cell clusters were depolymerized. Using mouse models, we validated the metastatic potential of tumor clusters was higher than single cells in vivo. Further analysis showed that higher expression of plakoglobin was able to promote activation of the PI3K/Akt/Bcl-2 pathway, which might protect the clusters from anoikis. Our data indicate that plakoglobin promotes tumor cluster formation in IMPC and downregulates apoptosis in the cell clusters through activation of PI3K/Akt/Bcl-2 signaling. These results provide a convincing rationale for the high metastatic propensity seen in IMPC.