Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Development of a Peripherally Restricted 5-HT2B Partial Agonist for Treatment of Pulmonary Arterial Hypertension.

Ligands for the serotonin 2B receptor (5-HT2B) have shown potential to treat pulmonary arterial hypertension in preclinical models but cannot be used in humans because of predicted off-target neurological effects. The aim of this study was to develop novel systemically restricted compounds targeting 5-HT2B. Here, we show that mice treated with VU6047534 had decreased RVSP compared with control treatment in both the prevention and intervention studies using Sugen-hypoxia. VU6047534 is a novel 5-HT2B partial agonist that is peripherally restricted and able to both prevent and treat Sugen-hypoxia-induced pulmonary arterial hypertension. We have synthesized and characterized a structurally novel series of 5-HT2B ligands with high potency and selectivity for the 5-HT2B receptor subtype. Next-generation 5-HT2B ligands with similar characteristics, and predicted to be systemically restricted in humans, are currently advancing to investigational new drug-enabling studies.

Myeloid Cell Derived IL1ß Contributes to Pulmonary Hypertension in HFpEF.

BACKGROUND: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are poorly understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. METHODS: Eight-week-old male and female C57BL/6J mice received either Nγ-nitro-L-arginine methyl ester and high-fat diet or control water and diet for 2, 5, and 12 weeks. The db/db mice were studied as a second model of HFpEF. Early pathways regulating PH were identified by bulk and single-cell RNA sequencing. Findings were confirmed by immunostain in lungs of mice or lung slides from clinically performed autopsies of patients with PH-HFpEF. ELISA was used to verify IL-1ß (interleukin-1 beta) in mouse lung, mouse plasma, and also human plasma from patients with PH-HFpEF obtained at the time of right heart catheterization. Clodronate liposomes and an anti-IL-1ß antibody were utilized to deplete macrophages and IL-1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF in mouse models. RESULTS: Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice developed PH, small vessel muscularization, and right heart dysfunction. Inflammation-related gene ontologies were overrepresented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling showed an increase in IL-1ß in mouse and human plasma. Finally, clodronate liposome treatment in mice prevented PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice, and IL-1ß depletion also attenuated PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice. CONCLUSIONS: We report a novel model for the study of PH and right heart remodeling in HFpEF, and we identify myeloid cell-derived IL-1ß as an important contributor to PH in HFpEF.

Activation of mTOR signaling in adult lung microvascular progenitor cells accelerates lung aging.

Reactivation and dysregulation of the mTOR signaling pathway are a hallmark of aging and chronic lung disease; however, the impact on microvascular progenitor cells (MVPCs), capillary angiostasis, and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue-resident MVPCs present in both adult mouse and human lungs using functional, lineage, and transcriptomic analyses. These studies link human and mouse MVPC-specific mTORC1 activation to decreased stemness, angiogenic potential, and disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation, these MVPCs adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function and microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.

Myeloid Cell Derived IL1ß Contributes to Pulmonary Vascular Remodeling in Heart Failure with Preserved Ejection Fraction.

Background: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are not well understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH in HFpEF, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. Methods: Eight week old male and female C57/BL6J mice were given either L-NAME and high fat diet (HFD) or control water/diet for 2,5, and 12 weeks. Bulk RNA sequencing and single cell RNA sequencing was performed to identify early and cell-specific pathways that might regulate pulmonary vascular remodeling in PH-HFpEF. Finally, clodronate liposome and IL1ß antibody treatments were utilized to deplete macrophages or IL1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF. Results: Mice given L-NAME/HFD developed PH, small vessel muscularization, and right heart dysfunction after 2 weeks of treatment. Inflammation-related gene ontologies were over-represented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling of mouse lung and plasma showed an increase in IL1ß, which was confirmed in plasma from patients with HFpEF. Single cell sequencing of mouse lungs also showed an increase in M1-like, pro-inflammatory populations of Ccr2+ monocytes and macrophages, and transcript expression of IL1ß was primarily restricted to myeloid-type cells. Finally, clodronate liposome treatment prevented the development of PH in L-NAME/HFD treated mice, and IL1ß antibody treatment also attenuated PH in L-NAME/HFD treated mice. Conclusions: Our study demonstrated that a well-accepted model of HFpEF recapitulates features of pulmonary vascular remodeling commonly seen in patients with HFpEF, and we identified myeloid cell derived IL1ß as an important contributor to PH in HFpEF.

Repetitive Sulfur Dioxide Exposure in Mice Models Post-Deployment Respiratory Syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than non-deployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the deployers in this cohort reported exposure to sulfur dioxide (SO 2 ), we developed a model of repetitive exposure to SO 2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular disease (PVD). Although abnormalities in small airways were not sufficient to alter lung mechanics, PVD was associated with the development of pulmonary hypertension and reduced exercise tolerance in SO 2 exposed mice. Further, we used pharmacologic and genetic approaches to demonstrate a critical role for oxidative stress and isolevuglandins in mediating PVD in this model. In summary, our results indicate that repetitive SO 2 exposure recapitulates many aspects of PDRS and that oxidative stress may mediate PVD in this model, which may be helpful for future mechanistic studies examining the relationship between inhaled irritants, PVD, and PDRS.

Molecular Function and Contribution of TBX4 in Development and Disease.

Over the past decade, recognition of the profound impact of the TBX4 (T-box 4) gene, which encodes a member of the evolutionarily conserved family of T-box-containing transcription factors, on respiratory diseases has emerged. The developmental importance of TBX4 is emphasized by the association of TBX4 variants with congenital disorders involving respiratory and skeletal structures; however, the exact role of TBX4 in human development remains incompletely understood. Here, we discuss the developmental, tissue-specific, and pathological TBX4 functions identified through human and animal studies and review the published TBX4 variants resulting in variable disease phenotypes. We also outline future research directions to fill the gaps in our understanding of TBX4 function and of how TBX4 disruption affects development.

l-Carnitine therapy improves right heart dysfunction through Cpt1-dependent fatty acid oxidation.

Pulmonary arterial hypertension (PAH) is a fatal vasculopathy that ultimately leads to elevated pulmonary pressure and death by right ventricular (RV) failure, which occurs in part due to decreased fatty acid oxidation and cytotoxic lipid accumulation. In this study, we tested the hypothesis that decreased fatty acid oxidation and increased lipid accumulation in the failing RV is driven, in part, by a relative carnitine deficiency. We then tested whether supplementation of l-carnitine can reverse lipotoxic RV failure through augmentation of fatty acid oxidation. In vivo in transgenic mice harboring a human BMPR2 mutation, l-carnitine supplementation reversed RV failure by increasing RV cardiac output, improving RV ejection fraction, and decreasing RV lipid accumulation through increased PPARγ expression and augmented fatty acid oxidation of long chain fatty acids. These findings were confirmed in a second model of pulmonary artery banding-induced RV dysfunction. In vitro, l-carnitine supplementation selectively increased fatty acid oxidation in mitochondria and decreased lipid accumulation through a Cpt1-dependent pathway. l-Carnitine supplementation improves right ventricular contractility in the stressed RV through augmentation of fatty acid oxidation and decreases lipid accumulation. Correction of carnitine deficiency through l-carnitine supplementation in PAH may reverse RV failure.

Experimental Approaches for Investigating Disulfide-Based Redox Relays in Cells.

Reversible oxidation of cysteine residues within proteins occurs naturally during normal cellular homeostasis and can increase during oxidative stress. Cysteine oxidation often leads to the formation of disulfide bonds, which can impact protein folding, stability, and function. Work in both prokaryotic and eukaryotic models over the past five decades has revealed several multiprotein systems that use thiol-dependent oxidoreductases to mediate disulfide bond reduction, formation, and/or rearrangement. Here, I provide an overview of how these systems operate to carry out disulfide exchange reactions in different cellular compartments, with a focus on their roles in maintaining redox homeostasis, transducing redox signals, and facilitating protein folding. Additionally, I review thiol-independent and thiol-dependent approaches for interrogating what proteins partner together in such disulfide-based redox relays. While the thiol-independent approaches rely either on predictive measures or standard procedures for monitoring protein-protein interactions, the thiol-dependent approaches include direct disulfide trapping methods as well as thiol-dependent chemical cross-linking. These strategies may prove useful in the systematic characterization of known and newly discovered disulfide relay mechanisms and redox switches involved in oxidant defense, protein folding, and cell signaling.

Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-ß (transforming growth factor-ß) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

Loss of talin in cardiac fibroblasts results in augmented ventricular cardiomyocyte hypertrophy in response to pressure overload.

Pressure overload of the heart is characterized by concentric hypertrophy and interstitial fibrosis. Cardiac fibroblasts (CFs) in the ventricular wall become activated during injury and synthesize and compact the extracellular matrix, which causes interstitial fibrosis and stiffening of the ventricular heart walls. Talin1 (Tln1) and Talin2 (Tln2) are mechanosensitive proteins that participate in focal adhesion transmission of signals from the extracellular environment to the actin cytoskeleton of CFs. The aim of the present study was to determine whether the removal of Tln1 and Tln2 from CFs would reduce interstitial fibrosis and cardiac hypertrophy. Twelve-week-old male and female Tln2-null (Tln2-/-) and Tln2-null, CF-specific Tln1 knockout (Tln2-/-;Tln1CF-/-) mice were given angiotensin-II (ANG II) (1.5 mg/kg/day) or saline through osmotic pumps for 8 wk. Cardiomyocyte area and measures of heart thickness were increased in the male ANG II-infused Tln2-/-;Tln1CF-/- mice, whereas there was no increase in interstitial fibrosis. Systolic blood pressure was increased in the female Tln2-/-;Tln1CF-/- mice after ANG II infusion compared with the Tln2-/- mice. However, there was no increase in cardiac hypertrophy in the Tln2-/-;Tln1CF-/- mice, which was seen in the Tln2-/- mice. Collectively, these data indicate that in male mice, the absence of Tln1 and Tln2 in CFs leads to cardiomyocyte hypertrophy in response to ANG II, whereas it results in a hypertrophy-resistant phenotype in female mice. These findings have important implications for the role of mechanosensitive proteins in CFs and their impact on cardiomyocyte function in the pathogenesis of hypertension and cardiac hypertrophy.NEW & NOTEWORTHY The role of talins has been previously studied in cardiomyocytes; however, these mechanotransductive proteins that are members of the focal adhesion complex have not been examined in cardiac fibroblasts previously. We hypothesized that loss of talins in cardiac fibroblasts would reduce interstitial fibrosis in the heart with a pressure overload model. However, we found that although loss of talins did not alter fibrosis, it did result in cardiomyocyte and ventricular hypertrophy.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

Broader than expected tolerance for substitutions in the WCGPCK catalytic motif of yeast thioredoxin 2.

Thioredoxins constitute a key class of oxidant defense enzymes that facilitate disulfide bond reduction in oxidized substrate proteins. While thioredoxin's WCGPCK active site motif is highly conserved in traditional model organisms, predicted thioredoxins from newly sequenced genomes show variability in this motif, making ascertaining which genes encode functional thioredoxins with robust activity a challenge. To address this problem, we generated a semi-saturation mutagenesis library of approximately 70 thioredoxin variants harboring mutations adjacent to their catalytic cysteines, making substitutions in the Saccharomyces cerevisiae thioredoxin Trx2. Using this library, we determined how such substitutions impact oxidant defense in yeast along with how they influence disulfide reduction and interaction with binding partners in vivo. The majority of thioredoxin variants screened rescued the slow growth phenotype that accompanies deletion of the yeast cytosolic thioredoxins; however, the ability of these mutant proteins to protect against H2O2-mediated toxicity, facilitate disulfide reduction, and interact with redox partners varied widely, depending on the site being mutated and the substitution made. We report that thioredoxin is less tolerant of substitutions at its conserved tryptophan and proline in the active site motif, while it is more amenable to substitutions at the conserved glycine and lysine. Our work highlights a noteworthy plasticity within the active site of this critical oxidant defense enzyme.

Urm1, not quite a ubiquitin-like modifier?

Ubiquitin related modifier 1 (Urm1) is a unique eukaryotic member of the ubiquitin-fold (UbF) protein family and conserved from yeast to humans. Urm1 is dual-functional, acting both as a sulfur carrier for thiolation of tRNA anticodons and as a protein modifier in a lysine-directed Ub-like conjugation also known as urmylation. Although Urm1 conjugation coincides with oxidative stress and targets proteins like 2-Cys peroxiredoxins from yeast (Ahp1) and fly (Prx5), it was unclear how urmylation proceeds molecularly and whether it is affected by the activity of these antioxidant enzymes. An in-depth study of Ahp1 urmylation in yeast from our laboratory (Brachmann et al., 2020) uncovered that promiscuous lysine target sites and specific redox requirements determine the Urm1 acceptor activity of the peroxiredoxin. The results clearly show that the dimer interface and the 2-Cys based redox-active centers of Ahp1 are affecting the Urm1 conjugation reaction. Together with in vivo assays demonstrating that high organic peroxide concentrations can prevent Ahp1 from being urmylated, Brachmann et al. provide insights into a potential link between Urm1 utilization and oxidant defense of cells. Here, we highlight these major findings and discuss wider implications with regards to an emerging link between Urm1 conjugation and redox biology. Moreover, from these studies we propose to redefine our perspective on Urm1 and the molecular nature of urmylation, a post-translational conjugation that may not be that ubiquitin-like after all.

KCNK3 Mutation Causes Altered Immune Function in Pulmonary Arterial Hypertension Patients and Mouse Models.

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we exposed kcnk3fl/fl animals to hypoxia, metabolic diet and low dose lipopolysaccharide (LPS) and performed molecular characterization of their tissue. We also used tissue samples from KCNK3 patients (skin fibroblast derived inducible pluripotent stem cells, blood, lungs, peripheral blood mononuclear cells) and performed microarray, immunohistochemistry (IHC) and mass cytometry time of flight (CyTOF) experiments. Although a hypoxic insult did not alter vascular tone in kcnk3fl/fl mice, RNASeq study of these lungs implied that inflammatory and metabolic factors were altered, and the follow-up diet study demonstrated a dysregulation of bone marrow cells in kcnk3fl/fl mice. Finally, a low dose LPS study clearly showed that inflammation could be a possible second hit driving PAH in kcnk3fl/fl mice. Multiplex, IHC and CyTOF immunophenotyping studies on human samples confirmed the mouse data and strongly indicated that cell mediated, and innate immune responses may drive PAH susceptibility in these patients. In conclusion, loss of function KCNK3 mutation alters various physiological processes from vascular tone to metabolic diet through inflammation. Our data suggests that altered circulating immune cells may drive PAH susceptibility in patients with KCNK3 mutation.

Expression of a Human Caveolin-1 Mutation in Mice Drives Inflammatory and Metabolic Defect-Associated Pulmonary Arterial Hypertension.

Background: In 2012, mutations in Cav1 were found to be the driving mutation in several cases of heritable pulmonary arterial hypertension (PAH). These mutations replaced the last 21 amino acids of Cav1 with a novel 22-amino-acid sequence. Because previously only Cav1 knockouts had been studied in the context of PAH, examining the in vivo effects of this novel mutation holds promise for new understanding of the role of Cav1 in disease etiology. Methods: The new 22 amino acids created by the human mutation were knocked into the native mouse Cav1 locus. The mice underwent hemodynamic, energy balance, and inflammatory measurements, both at baseline and after being stressed with either a metabolic or an inflammatory challenge [low-dose lipopolysaccharide (LPS)]. To metabolically challenge the mice, they were injected with streptozotocin (STZ) and fed a high-fat diet for 12 weeks. Results: Very little mutant protein was found in vivo (roughly 2% of wild-type by mass spectrometry), probably because of degradation after failure to traffic from the endoplasmic reticulum. The homozygous mutants developed a mild, low-penetrance PAH similar to that described previously in knockouts, and neither baseline nor metabolic nor inflammatory stress resulted in pressures above normal in heterozygous animals. The homozygous mutants had increased lean mass and worsened oral glucose tolerance, as previously described in knockouts. Novel findings include the preservation of Cav2 and accessory proteins in the liver and the kidney, while they are lost with homozygous Cav1 mutation in the lungs. We also found that the homozygous mutants had a significantly lower tolerance to voluntary spontaneous exercise than the wild-type mice, with the heterozygous mice at an intermediate level. The mutants also had higher circulating monocytes, with both heterozygous and homozygous animals having higher pulmonary MCP1 and MCP5 proteins. The heterozygous animals also lost weight at an LPS challenge level at which the wild-type mice continued to gain weight. Conclusions: The Cav1 mutation identified in human patients in 2012 is molecularly similar to a knockout of Cav1. It results in not only metabolic deficiencies and mild pulmonary hypertension, as expected, but also an inflammatory phenotype and reduced spontaneous exercise.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

A multifaceted investigation into molecular associations of chronic thromboembolic pulmonary hypertension pathogenesis.

PURPOSE: Chronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension and right ventricular dysfunction. Conditions such as thrombophilias, dysfibrinogenemias, and inflammatory states have been associated with chronic thromboembolic pulmonary hypertension, but molecular mechanisms underlying this disease are poorly understood. We sought to characterize the molecular and functional features associated with chronic thromboembolic pulmonary hypertension using a multifaceted approach. METHODS: We utilized functional assays to compare clot lysis times between chronic thromboembolic pulmonary hypertension patients and multiple controls. We then performed immunohistochemical characterization of tissue from chronic thromboembolic pulmonary hypertension, pulmonary arterial hypertension, and healthy controls, and examined RNA expression patterns of cultured lymphocytes and pulmonary arterial specimens. We then confirmed RNA expression changes using immunohistochemistry, immunofluorescence, and Western blotting in pulmonary arterial tissue. RESULTS: Clot lysis times in chronic thromboembolic pulmonary hypertension patients are similar to multiple controls. Chronic thromboembolic pulmonary hypertension endarterectomized tissue has reduced expression of both smooth muscle and endothelial cell markers. RNA expression profiles in pulmonary arteries and peripheral blood lymphocytes identified differences in RNA transcript levels related to inflammation and growth factor signaling, which we confirmed using immunohistochemistry. Gene expression data also suggested significant alterations in metabolic pathways, and immunofluorescence and Western blot experiments confirmed that unglycosylated CD36 and adiponectin expression were increased in chronic thromboembolic pulmonary hypertension versus controls. CONCLUSIONS: Our data do not support impaired clot lysis underlying chronic thromboembolic pulmonary hypertension, but did demonstrate distinct molecular patterns present both in peripheral blood and in pathologic specimens of chronic thromboembolic pulmonary hypertension patients suggesting that altered metabolism may play a role in chronic thromboembolic pulmonary hypertension pathogenesis.

Redox requirements for ubiquitin-like urmylation of Ahp1, a 2-Cys peroxiredoxin from yeast.

The yeast peroxiredoxin Ahp1, like related anti-oxidant enzymes in other species, undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Ahp1 assembles into a homodimer that detoxifies peroxides via forming intersubunit disulfides between peroxidatic and resolving cysteines that are subsequently reduced by the thioredoxin system. Although urmylation coincides with oxidative stress, it is unclear how this modification happens on a molecular level and whether it affects peroxiredoxin activity. Here, we report that thioredoxin mutants decrease Ahp1 urmylation in yeast and each subunit of the oxidized Ahp1 dimer is modified by Urm1 suggesting coupling of urmylation to dimerization. Consistently, Ahp1 mutants unable to form dimers, fail to be urmylated as do mutants that lack the peroxidatic cysteine. Moreover, Ahp1 urmylation involves at least two lysine residues close to the catalytic cysteines and can be prevented in yeast cells exposed to high organic peroxide concentrations. Our results elucidate redox requirements and molecular determinants critical for Ahp1 urmylation, thus providing insights into a potential link between oxidant defense and Urm1 utilization in cells.