Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFß-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor.

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Dysregulation of FOXO1 (Forkhead Box O1 Protein) Drives Calcification in Arterial Calcification due to Deficiency of CD73 and Is Present in Peripheral Artery Disease.

OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.

A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Furthermore, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

MOV10 Provides Antiviral Activity against RNA Viruses by Enhancing RIG-I-MAVS-Independent IFN Induction.

Moloney leukemia virus 10, homolog (MOV10) is an IFN-inducible RNA helicase, associated with small RNA-induced silencing. In this article, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive- and negative-strand RNA viruses by enhancing type I IFN induction. Using a number of genome-edited knockout human cells, we show that IFN regulatory factor 3-mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IFN regulatory factor 3-mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known retinoic acid-inducible gene I/mitochondrial antiviral-signaling protein-mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required inhibitor of κB kinase ε, not TANK-binding kinase 1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses an retinoic acid-inducible gene I-like receptor-independent pathway to enhance IFN response.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

Rapamycin increases murine lifespan but does not reduce mineral volume in the Matrix GLA Protein (MGP) knockout mouse model of medial arterial calcification.

Peripheral artery disease (PAD) is the narrowing of the arteries that carry blood to the lower extremities. PAD has been traditionally associated with atherosclerosis. However, recent studies have found that medial arterial calcification (MAC) is the primary cause of chronic limb ischemia below the knee. MAC involves calcification of the elastin fibers surrounding smooth muscle cells (SMCs) in arteries. Matrix GLA Protein (MGP) binds circulating calcium and inhibits vascular calcification. Mgp -/- mice develop severe MAC and die within 8 weeks of birth due to aortic rupture or heart failure. We previously discovered a rare genetic disease Arterial Calcification due to Deficiency in CD73 (ACDC) in which patients present with extensive MAC in their lower extremity arteries. Using a patient-specific induced pluripotent stem cell model we found that rapamycin inhibited calcification. Here we investigated whether rapamycin could reduce MAC in vivo using Mgp -/- mice as a model. Mgp +/+ and Mgp -/- mice received 5mg/kg rapamycin or vehicle. Calcification content was assessed via microCT, and vascular morphology and extracellular matrix content assessed histologically. Immunostaining and western blot analysis were used to examine SMC phenotypes and cellular functions. Rapamycin prolonged Mgp -/- mice lifespan, decreased mineral density in the arteries, and increased smooth muscle actin protein levels, however, calcification volume, vessel morphology, SMC proliferation, and autophagy flux were all unchanged. These findings suggest that rapamycin's effects in the Mgp -/- mouse are independent of the vascular phenotype.

Isolation of Human Primary Valve Cells for In vitro Disease Modeling.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Gene Expression Analysis in the Pneumococcus.

Bacterial cells modify their gene expression profiles throughout different stages of growth and in response to environmental cues. Analyses of gene expression across conditions reveal both conserved and condition-specific gene responses of bacteria to adapt to these dynamic conditions. In this chapter, we present a guide to pneumococcal RNA extraction for use in the NanoString nCounter platform. The nCounter is a highly effective method to measure gene expression of bacteria not only in a planktonic mode of growth but also in the presence of host cells where the RNA of interest represents only a small portion of the total material.

Gene Acquisition by a Distinct Phyletic Group within Streptococcus pneumoniae Promotes Adhesion to the Ocular Epithelium.

Streptococcus pneumoniae (pneumococcus) displays broad tissue tropism and infects multiple body sites in the human host. However, infections of the conjunctiva are limited to strains within a distinct phyletic group with multilocus sequence types ST448, ST344, ST1186, ST1270, and ST2315. In this study, we sequenced the genomes of six pneumococcal strains isolated from eye infections. The conjunctivitis isolates are grouped in a distinct phyletic group together with a subset of nasopharyngeal isolates. The keratitis (infection of the cornea) and endophthalmitis (infection of the vitreous body) isolates are grouped with the remainder of pneumococcal strains. Phenotypic characterization is consistent with morphological differences associated with the distinct phyletic group. Specifically, isolates from the distinct phyletic group form aggregates in planktonic cultures and chain-like structures in biofilms grown on abiotic surfaces. To begin to investigate the association between genotype and epidemiology, we focused on a predicted surface-exposed adhesin (SspB) encoded exclusively by this distinct phyletic group. Phylogenetic analysis of the gene encoding SspB in the context of a streptococcal species tree suggests that sspB was acquired by lateral gene transfer from Streptococcus suis. Furthermore, an sspB deletion mutant displays decreased adherence to cultured cells from the ocular epithelium compared to the isogenic wild-type and complemented strains. Together these findings suggest that acquisition of genes from outside the species has contributed to pneumococcal tissue tropism by enhancing the ability of a subset of strains to infect the ocular epithelium causing conjunctivitis. IMPORTANCE Changes in the gene content of pathogens can modify their ability to colonize and/or survive in different body sites in the human host. In this study, we investigate a gene acquisition event and its role in the pathogenesis of Streptococccus pneumoniae (pneumococcus). Our findings suggest that the gene encoding the predicted surface protein SspB has been transferred from Streptococcus suis (a distantly related streptococcal species) into a distinct set of pneumococcal strains. This group of strains distinguishes itself from the remainder of pneumococcal strains by extensive differences in genomic composition and by the ability to cause conjunctivitis. We find that the presence of sspB increases adherence of pneumococcus to the ocular epithelium. Thus, our data support the hypothesis that a subset of pneumococcal strains has gained genes from neighboring species that enhance their ability to colonize the epithelium of the eye, thus expanding into a new niche.