High Glucose Levels Promote Switch to Synthetic Vascular Smooth Muscle Cells via Lactate/GPR81.

Hyperglycemia, lipotoxicity, and insulin resistance are known to increase the secretion of extracellular matrix from cardiac fibroblasts as well as the activation of paracrine signaling from cardiomyocytes, immune cells, and vascular cells, which release fibroblast-activating mediators. However, their influences on vascular smooth muscle cells (vSMCs) have not been well examined. This study aimed to investigate whether contractile vascular vSMCs could develop a more synthetic phenotype in response to hyperglycemia. The results showed that contractile and synthetic vSMCs consumed high glucose in different ways. Lactate/GPR81 promotes the synthetic phenotype in vSMCs in response to high glucose levels. The stimulation of high glucose was associated with a significant increase in fibroblast-like features: synthetic vSMC marker expression, collagen 1 production, proliferation, and migration. GPR81 expression is higher in blood vessels in diabetic patients and in the high-glucose, high-lipid diet mouse. The results demonstrate that vSMCs assume a more synthetic phenotype when cultured in the presence of high glucose and, consequently, that the high glucose could trigger a vSMC-dependent cardiovascular disease mechanism in diabetes via lactate/GPR81.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

SFPQ and Its Isoform as Potential Biomarker for Non-Small-Cell Lung Cancer.

Cancer markers are measurable molecules in the blood or tissue that are produced by tumor cells or immune cells in response to cancer progression. They play an important role in clinical diagnosis, prognosis, and anti-drug monitoring. Although DNA, RNA, and even physical images have been used, proteins continue to be the most common marker. There are currently no specific markers for lung cancer. Metastatic lung cancer, particularly non-small-cell lung cancer (NSCLC), is one of the most common causes of death. SFPQ, YY1, RTN4, RICTOR, LARP6, and HELLS are expressed at higher levels in cells from NSCLC than in control or cells from inflammatory diseases. SFPQ shows the most difference between the three cell types. Furthermore, the cytoplasmic isoform of SFPQ is only found in advanced cancers. We have developed ELISAs to detect SFPQ and the long and short isoforms. Evidence has shown that the short isoform exists primarily in cancers. Furthermore, immunocytometry studies and IHC analysis have revealed that SFPQ levels are consistent with ELISA results. In addition, enhanced DNA methylation in the SFPQ gene may facilitate the SFPQ expression differences between control and cancer cells. Considering this, elevated SFPQ level and the isoform location could serve as a cancer diagnostic and prognostic marker.

IL-8 concurrently promotes idiopathic pulmonary fibrosis mesenchymal progenitor cell senescence and PD-L1 expression enabling escape from immune cell surveillance.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We discovered fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that display cell-autonomous fibrogenicity and drive fibrotic progression. In a study of the IPF MPC nuclear proteome, we identified DNA damage as one of the most altered functions in IPF MPCs. In prior work we found that IL-8 drives IPF MPC self-renewal. IL-8 can promote replicative stress and DNA damage and induce senescence through the CXCR2 receptor. We hypothesized that IL-8 promotes DNA damage-mediated senescence in IPF MPCs. We show that IL-8 induces DNA damage and promotes IPF MPC senescence. We discovered that IL-8 concurrently promotes senescence and upregulation of the programmed death ligand 1 (PD-L1) in a CXCR2-dependent manner. Disruption of programmed cell death protein-1 (PD-1)-PD-L1 interaction promotes natural killer (NK) cell killing of IPF MPCs in vitro and arrests IPF MPC-mediated experimental lung fibrosis in vivo. Immunohistochemical (IHC) analysis of IPF lung tissue identified PD-L1-expressing IPF MPCs codistributing with NK cells and ß-galactosidase-positive cells. Our data indicate that IL-8 simultaneously promotes IPF MPC DNA damage-induced senescence and high PD-L1 expression, enabling IPF MPCs to elude immune cell-targeted removal. Disruption of PD-1-PD-L1 interaction may limit IPF MPC-mediated fibrotic progression.NEW & NOTEWORTHY Here we show that IL-8 concurrently promotes senescence and upregulation of PD-L1 in IPF MPCs. IHC analysis identifies the presence of senescent IPF MPCs intermingled with NK cells in the fibroblastic focus, suggesting that senescent MPCs elude immune cell surveillance. We demonstrate that disruption of PD-1/PD-L1 interaction promotes NK cell killing of IPF MPCs and arrests IPF MPC-mediated experimental lung fibrosis. Disruption of PD-1/PD-L1 interaction may be one means to limit fibrotic progression.

Hypoxia enhances IPF mesenchymal progenitor cell fibrogenicity via the lactate/GPR81/HIF1α pathway.

Hypoxia is a sentinel feature of idiopathic pulmonary fibrosis (IPF). The IPF microenvironment contains high lactate levels, and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) that drive fibrosis in the lungs of patients with IPF. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, and this increase in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrates IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knockdown of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.

SFPQ Promotes Lung Cancer Malignancy via Regulation of CD44 v6 Expression.

Mesenchymal stem cells (MSCs) contribute to tumor pathogenesis and elicit antitumor immune responses in tumor microenvironments. Nuclear proteins might be the main players in these processes. In the current study, combining spatial proteomics with ingenuity pathway analysis (IPA) in lung non-small cell (NSC) cancer MSCs, we identify a key nuclear protein regulator, SFPQ (Splicing Factor Proline and Glutamine Rich), which is overexpressed in lung cancer MSCs and functions to promote MSCs proliferation, chemical resistance, and invasion. Mechanistically, the knockdown of SFPQ reduces CD44v6 expression to inhibit lung cancer MSCs stemness, proliferation in vitro, and metastasis in vivo. The data indicates that SFPQ may be a potential therapeutic target for limiting growth, chemotherapy resistance, and metastasis of lung cancer.

Proteomic analysis of the IPF mesenchymal progenitor cell nuclear proteome identifies abnormalities in key nodal proteins that underlie their fibrogenic phenotype.

IPF is a progressive fibrotic lung disease whose pathogenesis remains incompletely understood. We have previously discovered pathologic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients. IPF MPCs display a distinct transcriptome and create sustained interstitial fibrosis in immune deficient mice. However, the precise pathologic alterations responsible for this fibrotic phenotype remain to be uncovered. Quantitative mass spectrometry and interactomics is a powerful tool that can define protein alterations in specific subcellular compartments that can be implemented to understand disease pathogenesis. We employed quantitative mass spectrometry and interactomics to define protein alterations in the nuclear compartment of IPF MPCs compared to control MPCs. We identified increased nuclear levels of PARP1, CDK1, and BACH1. Interactomics implicated PARP1, CDK1, and BACH1 as key hub proteins in the DNA damage/repair, differentiation, and apoptosis signaling pathways respectively. Loss of function and inhibitor studies demonstrated important roles for PARP1 in DNA damage/repair, CDK1 in regulating IPF MPC stemness and self-renewal, and BACH1 in regulating IPF MPC viability. Our quantitative mass spectrometry studies combined with interactomic analysis uncovered key roles for nuclear PARP1, CDK1, and BACH1 in regulating IPF MPC fibrogenicity.

A CD44/Brg1 nuclear complex confers mesenchymal progenitor cells with enhanced fibrogenicity in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We previously identified fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of patients with IPF who serve as drivers of progressive fibrosis. Recent single-cell RNA sequencing work revealed that IPF MPCs with the highest transcriptomic network entropy differ the most from control MPCs and that increased CD44 was a marker of these IPF MPCs. We hypothesize that IPF MPCs with high CD44 (CD44hi) expression will display enhanced fibrogenicity. We demonstrate that CD44-expressing MPCs are present at the periphery of the IPF fibroblastic focus, placing them in regions of active fibrogenesis. In a humanized mouse xenograft model, CD44hi IPF MPCs are more fibrogenic than CD44lo IPF MPCs, and knockdown of CD44 diminishes their fibrogenicity. CD44hi IPF MPCs display increased expression of pluripotency markers and enhanced self-renewal compared with CD44lo IPF MPCs, properties potentiated by IL-8. The mechanism involves the accumulation of CD44 within the nucleus, where it associates with the chromatin modulator protein Brahma-related gene 1 (Brg1) and the zinc finger E-box binding homeobox 1 (Zeb1) transcription factor. This CD44/Brg1/Zeb1 nuclear protein complex targets the Sox2 gene, promoting its upregulation and self-renewal. Our data implicate CD44 interaction with the epigenetic modulator protein Brg1 in conveying IPF MPCs with cell-autonomous fibrogenicity.

Hyaluronan/CD44 axis regulates S100A4-mediated mesenchymal progenitor cell fibrogenicity in idiopathic pulmonary fibrosis.

Despite modest improvement in patient outcomes from recent advances in pharmacotherapy targeting fibrogenic signaling pathways, idiopathic pulmonary fibrosis (IPF) remains a major unsolved clinical problem. One reason for this is that available antifibrotic agents slow down but do not arrest fibrotic progression. To arrest fibrotic progression, its obligatory drivers need to be identified. We previously discovered that fibrogenic mesenchymal progenitor cells (MPCs) are key drivers of fibrotic progression in IPF, serving as cells of origin for disease-mediating myofibroblasts. IPF MPCs have high levels of nuclear S100A4, which interacts with the proteasome to promote p53 degradation and self-renewal. However, the mechanism underlying S100A4 accumulation in the nucleus of IPF MPCs remains unknown. Here we show that hyaluronan (HA) is present in the fibroblastic focus together with CD44-expressing MPCs and that ligation of CD44 by HA triggers S100A4 nuclear translocation to support IPF MPC self-renewal. The mechanism involves HA-mediated formation of a CD44/S100A4/transportin 1 complex, which promotes S100A4 nuclear import. In a humanized mouse model of pulmonary fibrosis, IPF MPC fibrogenicity was significantly attenuated by 1) knockdown of CD44 or 2) introduction of an S100A4 mutant construct that prevents S100A4 nuclear import. These data indicate that signaling through the HA/CD44/S100A4 axis is an integral component of IPF MPC fibrogenicity.

Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory.

In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

Ceftolozane-tazobactam and ceftazidime-avibactam activity against ß-lactam-resistant Pseudomonas aeruginosa and extended-spectrum ß-lactamase-producing Enterobacterales clinical isolates from U.S. medical centres.

BACKGROUND: Despite availability of ceftolozane-tazobactam (C/T) and ceftazidime-avibactam (CZA) for several years, the individual spectrum of activity of each agent may not be widely known. We compared the activity of C/T and CZA against convenience samples of 119 extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales and 60 ß-lactam-resistant Pseudomonas aeruginosa clinical isolates collected from three U.S. institutions. METHODS: Minimal inhibitory concentrations (MICs) for C/T and CZA were determined by broth microdilution. Molecular identification of nine ß-lactamase gene targets was conducted for Enterobacterales and P. aeruginosa isolates with increased MICs to C/T or CZA. RESULTS: More than 90% of Enterobacterales isolates demonstrated susceptibility to both C/T and CZA, in contrast to the other traditional ß-lactam agents tested, which were much less active. The MIC50/90 values were nearly equivalent between agents. The most common ß-lactamase genes identified in Enterobacterales isolates with MIC values ≥2 mg/L were the CTX-M-1 group (85%) and CMY-2-like (23%) ß-lactamases. Both agents were active against >80% of ß-lactam-resistant P. aeruginosa isolates tested, most of which had oprD mutations identified. One P. aeruginosa isolate was positive for a Klebsiella pneumoniae carbapenemase-type gene but remained meropenem-susceptible. The MIC50 values were four-fold lower in favour of C/T (1 mg/L vs. 4 mg/L) against P. aeruginosa. CONCLUSIONS: Our data suggest that either agent may be a reasonable choice for centres with a high proportion of ESBL producers; however, C/T may have improved activity against P. aeruginosa and may be preferred in institutions with a higher frequency of resistant pseudomonal isolates.

Agmatine accumulation by Pseudomonas aeruginosa clinical isolates confers antibiotic tolerance and dampens host inflammation.

PURPOSE: In the cystic fibrosis (CF) airways, Pseudomonas aeruginosa undergoes diverse physiological changes in response to inflammation, antibiotic pressure, oxidative stress and a dynamic bioavailable nutrient pool. These include loss-of-function mutations that result in reduced virulence, altered metabolism and other phenotypes that are thought to confer a selective advantage for long-term persistence. Recently, clinical isolates of P. aeruginosa that hyperproduce agmatine (decarboxylated arginine) were cultured from individuals with CF. Sputum concentrations of this metabolite were also shown to correlate with disease severity. This raised the question of whether agmatine accumulation might also confer a selective advantage for P. aeruginosa during chronic colonization of the lung. METHODOLOGY AND RESULTS: We screened a library of P. aeruginosa CF clinical isolates and found that ~5 % of subjects harboured isolates with an agmatine hyperproducing phenotype. Agmatine accumulation was a direct result of mutations in aguA, encoding the arginine deiminase that catalyses the conversion of agmatine into various polyamines. We also found that agmatine hyperproducing isolates (aguA-) had increased tolerance to the cationic antibiotics gentamicin, tobramycin and colistin relative to their chromosomally complemented strains (aguA+). Finally, we revealed that agmatine diminishes IL-8 production by airway epithelial cells in response to bacterial infection, with a consequent decrease in neutrophil recruitment to the murine airways in an acute pneumonia model. CONCLUSION: These data highlight a potential new role for bacterial-derived agmatine that may have important consequences for the long-term persistence of P. aeruginosa in the CF airways.

Dicer1 Deficiency in the Idiopathic Pulmonary Fibrosis Fibroblastic Focus Promotes Fibrosis by Suppressing MicroRNA Biogenesis.

RATIONALE: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. OBJECTIVES: To determine how IPF-ECM negatively regulates miR-29. METHODS: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. MEASUREMENTS AND MAIN RESULTS: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. CONCLUSIONS: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

IL-8 mediates idiopathic pulmonary fibrosis mesenchymal progenitor cell fibrogenicity.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease, but the mechanisms driving progression remain incompletely defined. We previously reported that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs), which serve as a cell of origin for IPF fibroblasts. Proliferating IPF MPCs are located at the periphery of fibroblastic foci in an active cellular front at the interface between the myofibroblast-rich focus core and adjacent normal alveolar structures. Among a large set of genes that distinguish IPF MPCs from their control counterparts, we identified IL-8 as a candidate mediator of IPF MPC fibrogenicity and driver of fibrotic progression. IPF MPCs and their progeny displayed increased steady-state levels of IL-8 and its cognate receptor CXCR1 and secreted more IL-8 than did controls. IL-8 functioned in an autocrine manner promoting IPF MPC self-renewal and the proliferation and motility of IPF MPC progeny. Secreted IL-8 also functioned in a paracrine manner stimulating macrophage migration. Analysis of IPF lung tissue demonstrated codistribution of IPF MPCs with activated macrophages in the active cellular front of the fibroblastic focus. These findings indicate that IPF MPC-derived IL-8 is capable of expanding the mesenchymal cell population and recruiting activated macrophages cells to actively evolving fibrotic lesions.

Lactate Promotes Synthetic Phenotype in Vascular Smooth Muscle Cells.

RATIONALE: The phenotypes of vascular smooth muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood. OBJECTIVE: To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs. METHODS AND RESULTS: Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L-) medium. Compared with the L- medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters and were generally attenuated both by the blockade of monocarboxylic acid transporter activity and by transfection with iRNA for NDRG (N-myc downstream regulated gene). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and NDRG expression were greater in the ischemic zone than in nonischemic tissues. CONCLUSIONS: These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.

Calcium-binding protein S100A4 confers mesenchymal progenitor cell fibrogenicity in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a prevalence of 1 million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli and leads to death by asphyxiation. We previously discovered that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs) that serve as a cell of origin for disease-mediating myofibroblasts. In a prior genomewide transcriptional analysis, we found that IPF MPCs displayed increased expression of S100 calcium-binding A4 (S100A4), a protein linked to cancer cell proliferation and invasiveness. Here, we have examined whether S100A4 mediates MPC fibrogenicity. Ex vivo analysis revealed that IPF MPCs had increased levels of nuclear S100A4, which interacts with L-isoaspartyl methyltransferase to promote p53 degradation and MPC self-renewal. In vivo, injection of human IPF MPCs converted a self-limited bleomycin-induced mouse model of lung fibrosis to a model of persistent fibrosis in an S100A4-dependent manner. S100A4 gain of function was sufficient to confer fibrotic properties to non-IPF MPCs. In IPF tissue, fibroblastic foci contained cells expressing Ki67 and the MPC markers SSEA4 and S100A4. The expression colocalized in an interface region between myofibroblasts in the focus core and normal alveolar structures, defining this region as an active fibrotic front. Our findings indicate that IPF MPCs are intrinsically fibrogenic and that S100A4 confers MPCs with fibrogenicity.

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1% (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 µM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.

The arginine decarboxylase pathways of host and pathogen interact to impact inflammatory pathways in the lung.

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.

Development of a Pseudomonas aeruginosa Agmatine Biosensor.

Agmatine, decarboxylated arginine, is an important intermediary in polyamine production for many prokaryotes, but serves higher functions in eukaryotes such as nitric oxide inhibition and roles in neurotransmission. Pseudomonas aeruginosa relies on the arginine decarboxylase and agmatine deiminase pathways to convert arginine into putrescine. One of the two known agmatine deiminase operons, aguBA, contains an agmatine sensitive TetR promoter controlled by AguR. We have discovered that this promoter element can produce a titratable induction of its gene products in response to agmatine, and utilized this discovery to make a luminescent agmatine biosensor in P. aeruginosa. The genome of the P. aeruginosa lab strain UCBPP-PA14 was altered to remove both its ability to synthesize or destroy agmatine, and insertion of the luminescent reporter construct allows it to produce light in proportion to the amount of exogenous agmatine applied from ~100 nM to 1mM. Furthermore it does not respond to related compounds including arginine or putrescine. To demonstrate potential applications the biosensor was used to detect agmatine in spent supernatants, to monitor the development of arginine decarboxylase over time, and to detect agmatine in the spinal cords of live mice.