The Involvement of Unfolded Protein Response in the Mechanism of Nitrogen Mustard-Induced Ocular Toxicity.

Nitrogen mustard (NM) is a known surrogate of sulfur mustard, a chemical-warfare agent that causes a wide range of ocular symptoms, from a permanent reduction in visual acuity to blindness upon exposure. Although it has been proposed that the two blistering agents have a similar mechanism of toxicity, the mode of NM-induced cell death in ocular tissue has not been fully explored. Therefore, we hypothesized that direct ocular exposure to NM in mice leads to retinal tissue injury through chronic activation of the unfolded protein response (UPR) PERK arm in corneal cells and VEGF secretion, eventually causing cell death. We topically applied NM directly to mice to analyze ocular and retinal tissues at 2 weeks postexposure. A dramatic decline in retinal function, measured by scotopic and photopic electroretinogram responses, was detected in the mice. This decline was associated with enhanced TUNEL staining in both corneal and retinal tissues. In addition, exposure of corneal cells to NM revealed 228 differentially and exclusively expressed proteins primarily associated with the UPR, ferroptosis, and necroptosis. Moreover, these cells exhibited activation of the UPR PERK arm and an increase in VEGF secretion. Enhancement of VEGF staining was later observed in the corneas of the exposed mice. Therefore, our data indicated that the mechanism of NM-induced ocular toxicity should be carefully examined and that future research should identify a signaling molecule transmitted via a prodeath pathway from the cornea to the retina. SIGNIFICANCE STATEMENT: This study demonstrated that NM topical exposure in mice results in dramatic decline in retinal function associated with enhanced TUNEL staining in both corneal and retinal tissues. We also found that the NM treatment of corneal cells resulted in 228 differentially and exclusively expressed proteins primarily associated with ferroptosis. Moreover, these cells manifest the UPR PERK activation and an increase in VEGF secretion. The latter was also found in the corneas of the cexposed mice.

Hemopexin reverses activation of lung eIF2α and decreases mitochondrial injury in chlorine-exposed mice.

We assessed the mechanisms by which nonencapsulated heme, released in the plasma of mice after exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult male and female C57BL/6 mice to Cl2 (500 ppm for 30 min), returned them to room air, and injected them intramuscularly with either human hemopexin (hHPX; 5 µg/g BW in 50-µL saline) or vehicle at 1 h post-exposure. Upon return to room air, Cl2-exposed mice, injected with vehicle, developed respiratory acidosis, increased concentrations of plasma proteins in the alveolar space, lung mitochondrial DNA injury, increased levels of free plasma heme, and major alterations of their lung proteome. hHPX injection mice mitigated the onset and development of lung and mitochondrial injury and the increase of plasma heme, reversed the Cl2-induced changes in 83 of 237 proteins in the lung proteome at 24 h post-exposure, and improved survival at 15 days post-exposure. Systems biology analysis of the lung global proteomics data showed that hHPX reversed changes in a number of key pathways including elF2 signaling, verified by Western blotting measurements. Recombinant human hemopexin, generated in tobacco plants, injected at 1 h post-Cl2 exposure, was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and the therapeutic effects of hemopexin.NEW & NOTEWORTHY Herein, we demonstrate that exposure of mice to chlorine gas causes significant changes in the lung proteome 24 h post-exposure. Systems biology analysis of the proteomic data is consistent with damage to mitochondria and activation of eIF2, the master regulator of transcription and protein translation. Post-exposure injection of hemopexin, which scavenges free heme, attenuated mtDNA injury, eIF2α phosphorylation, decreased lung injury, and increased survival.

Proteomic analysis of diabetic retinas.

Introduction: As a metabolic disease, diabetes often leads to health complications such as heart failure, nephropathy, neurological disorders, and vision loss. Diabetic retinopathy (DR) affects as many as 100 million people worldwide. The mechanism of DR is complex and known to impact both neural and vascular components in the retina. While recent advances in the field have identified major cellular signaling contributing to DR pathogenesis, little has been reported on the protein post-translational modifications (PTM) - known to define protein localization, function, and activity - in the diabetic retina overall. Protein glycosylation is the enzymatic addition of carbohydrates to proteins, which can influence many protein attributes including folding, stability, function, and subcellular localization. O-linked glycosylation is the addition of sugars to an oxygen atom in amino acids with a free oxygen atom in their side chain (i.e., threonine, serine). To date, more than 100 congenital disorders of glycosylation have been described. However, no studies have identified the retinal O-linked glycoproteome in health or disease. With a critical need to expedite the discovery of PTMomics in diabetic retinas, we identified both global changes in protein levels and the retinal O-glycoproteome of control and diabetic mice. Methods: We used liquid chromatography/mass spectrometry-based proteomics and high throughput screening to identify proteins differentially expressed and proteins differentially O-glycosylated in the retinas of wildtype and diabetic mice. Results: Changes in both global expression levels of proteins and proteins differentially glycosylated in the retinas of wild-type and diabetic mice have been identified. We provide evidence that diabetes shifts both global expression levels and O-glycosylation of metabolic and synaptic proteins in the retina. Discussion: Here we report changes in the retinal proteome of diabetic mice. We highlight alterations in global proteins involved in metabolic processes, maintaining cellular structure, trafficking, and neuronal processes. We then showed changes in O-linked glycosylation of individual proteins in the diabetic retina.

Early-adulthood spike in protein translation drives aging via juvenile hormone/germline signaling.

Protein translation (PT) declines with age in invertebrates, rodents, and humans. It has been assumed that elevated PT at young ages is beneficial to health and PT ends up dropping as a passive byproduct of aging. In Drosophila, we show that a transient elevation in PT during early-adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later-life. Blocking the early-life PT elevation robustly improves life-/health-span and prevents age-related protein aggregation, whereas transiently inducing an early-life PT surge in long-lived fly strains abolishes their longevity/proteostasis benefits. The early-life PT elevation triggers proteostatic dysfunction, silences stress responses, and drives age-related functional decline via juvenile hormone-lipid transfer protein axis and germline signaling. Our findings suggest that PT is adaptively suppressed after early-adulthood, alleviating later-life proteostatic burden, slowing down age-related functional decline, and improving lifespan. Our work provides a theoretical framework for understanding how lifetime PT dynamics shape future aging trajectories.

Hemopexin Reverses Activation of Lung eIF2a and Decreases Mitochondrial Injury in Chlorine Exposed Mice.

We assessed the mechanisms by which non-encapsulated heme, released in the plasma of mice post exposure to chlorine (Cl 2 ) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult C57BL/6 male and female to Cl 2 (500 ppm for 30 min) in environmental chambers and returned them to room air and injected them intramuscularly with a single dose of human hemopexin (hHPX; 5 µg/ g BW), the most efficient scavenger of heme, 30-60 min post exposure. Concentrations of hHPX in plasma of air and Cl 2 exposed mice were 9081±900 vs. 1879± 293 at 6 h and 2966±463 vs. 1555±250 at 50 h post injection (ng/ml; X±1 SEM=3; p<0.01). Cl 2 exposed mice developed progressive acute lung injury post exposure characterized by increased concentrations of plasma heme, marked inflammatory response, respiratory acidosis and increased concentrations of plasma proteins in the alveolar space. Injection of hHPX decreased the onset of acute lung injury at 24 h post exposure; mean survival, for the saline and hHPX groups were 40 vs. 80% (P<0.001) at 15 d post exposure. Non-supervised global proteomics analysis of mouse lungs at 24 h post exposure, revealed the upregulation of 92 and downregulation of 145 lung proteins. Injection of hHPX at one h post exposure moderated the Cl 2 induced changes in eighty-three of these 237 lung proteins. System biology analysis of the global proteomics data showed that hHPX reversed changes in mitochondrial dysfunction and elF2 and integrin signaling. Western blot analysis of lung tissue showed significant increase of phosphorylated elF2 at 24 h post exposure in vehicle treated mice but normal levels in those injected with hHPX. Similarly, RT-PCR analysis of lung tissue showed that hHPX reversed the onset of mtDNA lesions. A form of recombinant human hemopexin generated in tobacco plants was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl 2 results in acute lung injury and to the therapeutic effects of hemopexin.

Effects of heterozygous deletion of autism-related gene Cullin-3 in mice.

Autism Spectrum Disorder (ASD) is a developmental disorder in which children display repetitive behavior, restricted range of interests, and atypical social interaction and communication. CUL3, coding for a Cullin family scaffold protein mediating assembly of ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors, has been identified as a high-risk gene for autism. Although complete knockout of Cul3 results in embryonic lethality, Cul3 heterozygous mice have reduced CUL3 protein, demonstrate comparable body weight, and display minimal behavioral differences including decreased spatial object recognition memory. In measures of reciprocal social interaction, Cul3 heterozygous mice behaved similarly to their wild-type littermates. In area CA1 of hippocampus, reduction of Cul3 significantly increased mEPSC frequency but not amplitude nor baseline evoked synaptic transmission or paired-pulse ratio. Sholl and spine analysis data suggest there is a small yet significant difference in CA1 pyramidal neuron dendritic branching and stubby spine density. Unbiased proteomic analysis of Cul3 heterozygous brain tissue revealed dysregulation of various cytoskeletal organization proteins, among others. Overall, our results suggest that Cul3 heterozygous deletion impairs spatial object recognition memory, alters cytoskeletal organization proteins, but does not cause major hippocampal neuronal morphology, functional, or behavioral abnormalities in adult global Cul3 heterozygous mice.

Impact of early pericardial fluid chymase activation after cardiac surgery.

Introduction: Chymase is a highly destructive serine protease rapidly neutralized in the circulation by protease inhibitors. Here we test whether pericardial fluid (PCF) chymase activation and other inflammatory biomarkers determine intensive care unit length of stay, and explore mechanisms of chymase delivery by extracellular vesicles to the heart. Methods: PCF was collected from adult patients (17 on-pump; 13 off-pump) 4 h after cardiac surgery. Extracellular vesicles (EVs) containing chymase were injected into Sprague-Dawley rats to test for their ability to deliver chymase to the heart. Results: The mean intensive care unit (ICU) stay and mean total length of stay was 2.17 ± 3.8 days and 6.41 ± 1.3 days respectively. Chymase activity and 32 inflammatory markers did not differ in on-pump vs. off-pump cardiac surgery. Society of Thoracic Surgeons Predicted Risk of Morbidity and Mortality Score (STS-PROM), 4-hour post-surgery PCF chymase activity and C-X-C motif chemokine ligand 6 (CXCL6) were all independent predictors of ICU and total hospital length of stay by univariate analysis. Mass spectrometry of baseline PCF shows the presence of serine protease inhibitors that neutralize chymase activity. The compartmentalization of chymase within and on the surface of PCF EVs was visualized by immunogold labeling and transmission electron microscopy. A chymase inhibitor prevented EV chymase activity (0.28 fmol/mg/min vs. 14.14 fmol/mg/min). Intravenous injection of PCF EVs obtained 24 h after surgery into Sprague Dawley rats shows diffuse human chymase uptake in the heart with extensive cardiomyocyte damage 4 h after injection. Discussion: Early postoperative PCF chymase activation underscores its potential role in cardiac damage soon after on- or off-pump cardiac surgery. In addition, chymase in extracellular vesicles provides a protected delivery mechanism from neutralization by circulating serine protease inhibitors.

Influenza A-induced cystic fibrosis transmembrane conductance regulator dysfunction increases susceptibility to Streptococcus pneumoniae.

Influenza A virus (IAV) infection is commonly complicated by secondary bacterial infections that lead to increased morbidity and mortality. Our recent work demonstrates that IAV disrupts airway homeostasis, leading to airway pathophysiology resembling cystic fibrosis disease through diminished cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we use human airway organotypic cultures to investigate how IAV alters the airway microenvironment to increase susceptibility to secondary infection with Streptococcus pneumoniae (Spn). We observed that IAV-induced CFTR dysfunction and airway surface liquid acidification is central to increasing susceptibility to Spn. Additionally, we observed that IAV induced profound transcriptional changes in the airway epithelium and proteomic changes in the airway surface liquid in both CFTR-dependent and -independent manners. These changes correspond to multiple diminished host defense pathways and altered airway epithelial function. Collectively, these findings highlight both the importance of CFTR function during infectious challenge and demonstrate a central role for the lung epithelium in secondary bacterial infections following IAV.

Different-sized extracellular vesicles derived from stored red blood cells package diverse cargoes and cause distinct cellular effects.

BACKGROUND: The formation of extracellular vesicles (EVs) occurs during cold storage of RBCs. Transfusion of EVs may contribute to adverse responses in recipients receiving RBCs. However, EVs are poorly characterized with limited data on whether distinct vesicles are formed, their composition, and potential biological effects. STUDY DESIGN AND METHODS: Stored RBC-derived EVs were purified using protocols that separate larger microvesicle-like EVs (LEVs) from smaller exosome-like vesicles (SEVs). Vesicles were analyzed by electron microscopy, content of hemoglobin, heme, and proteins (by mass spectrometry), and the potential to mediate lipid peroxidation and endothelial cell permeability in vitro. RESULTS: SEVs were characterized by having an electron-dense double membrane whereas LEVs had more uniform electron density across the particles. No differences in hemoglobin nor heme levels per particle were observed, however, due to smaller volumes, SEVs had higher concentrations of oxyHb and heme. Both particles contained antioxidant proteins peroxiredoxin-2 and copper/zinc superoxide dismutase, these were present in higher molecular weight fractions in SEVs suggesting either oxidized proteins are preferentially packaged into smaller vesicles and/or that the environment associated with SEVs is more pro-oxidative. Furthermore, total glutathione (GSH + GSSG) levels were lower in SEVs. Both EVs mediated oxidation of liposomes that were prevented by hemopexin, identifying heme as the pro-oxidant effector. Addition of SEVs, but not LEVs, induced endothelial permeability in a process also prevented by hemopexin. CONCLUSION: These data show that distinct EVs are formed during cold storage of RBCs with smaller particles being more likely to mediate pro-oxidant and inflammatory effects associated with heme.

The SARS-CoV-2 spike S1 protein induces global proteomic changes in ATII-like rat L2 cells that are attenuated by hyaluronan.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants, COVID-19 has led to 651,918,402 confirmed cases and 6,656,601 deaths worldwide (as of December 27, 2022; https://covid19.who.int/). Despite advances in our understanding of COVID-19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in the proteome of alveolar type II (ATII)-like rat L2 cells that lack ACE2 receptors. Systems biology analysis revealed that the S1-induced proteomics changes were associated with three significant network hubs: E2F1, CREB1/RelA, and ROCK2/RhoA. We also found that pretreatment of L2 cells with high molecular weight hyaluronan (HMW-HA) greatly attenuated the S1 effects on the proteome. Western blotting analysis and cell cycle measurements confirmed the S1 upregulation of E2F1 and ROCK2/RhoA in L2 cells and the protective effects of HMW-HA. Taken as a whole, our studies revealed profound and novel biological changes that contribute to our current understanding of both S1 and hyaluronan biology. These data show that the S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV-2-induced cell injury.

Plasma Exosome Hemoglobin Released During Surgery Is Associated With Cardiac Injury in Animal Model.

BACKGROUND: Patients with valvular heart disease require cardiopulmonary bypass and cardiac arrest. Here, we test the hypothesis that exosomal hemoglobin formed during cardiopulmonary bypass mediates acute cardiac injury in humans and in an animal model system. METHODS: Plasma exosomes were collected from arterial blood at baseline and 30 minutes after aortic cross-clamp release in 20 patients with primary mitral regurgitation and 7 with aortic stenosis. These exosomes were injected into Sprague-Dawley rats and studied at multiple times up to 30 days. Tissue was examined by hematoxylin and eosin stain, immunohistochemistry, transmission electron microscopy, and brain natriuretic peptide. RESULTS: Troponin I levels increased from 36 ± 88 ng/L to 3622 ± 3054 ng/L and correlated with exosome hemoglobin content (Spearman r = 0.7136, < .0001, n = 24). Injection of exosomes isolated 30 minutes after cross-clamp release into Sprague-Dawley rats resulted in cardiomyocyte myofibrillar loss at 3 days. Transmission electron microscopy demonstrated accumulation of electron dense particles of ferritin within cardiomyocytes, in the interstitial space, and within exosomes. At 21 days after injection, there was myofibrillar and myosin breakdown, interstitial fibrosis, elevated brain natriuretic peptide, and left ventricle diastolic dysfunction measured by echocardiography/Doppler. Pericardial fluid exosomal hemoglobin content is fourfold higher than simultaneous plasma exosome hemoglobin, suggesting a cardiac source of exosomal hemoglobin. CONCLUSIONS: Red blood cell and cardiac-derived exosomal hemoglobin may be involved in myocardial injury during cardiopulmonary bypass in patients with valvular heart disease.

Effects of an exogenous ketone ester using multi-omics in skeletal muscle of aging C57BL/6J male mice.

Exogenous ketone ester supplementation provides a means to increase circulating ketone concentrations without the dietary challenges imposed by ketogenic diets. Our group has shown that oral R,S-1,3, butanediol diacetoacetate (BD-AcAc2) consumption results in body weight loss or maintenance with moderate increases in circulating ketones. We have previously shown a diet consisting of 25% BD-AcAc2 can maintain lean body mass (LBM) and induce fat mass (FM) loss in young, healthy male mice, but the underlying mechanisms are still unknown. Therefore, the purpose of this study was to determine if a diet consisting of 25% BD-AcAc2 (ketone ester, KE) would alter body composition, transcriptional regulation, the proteome, and the lipidome of skeletal muscle in aged mice. We hypothesized that the KE group would remain weight stable with improvements in body composition compared to controls, resulting in a healthy aging phenotype. Male C57BL/6J mice (n = 16) were purchased from Jackson Laboratories at 72 weeks of age. After 1 week of acclimation, mice were weighed and randomly assigned to one of two groups (n = 8 per group): control (CON) or KE. A significant group by time interaction was observed for body weight (P < 0.001), with KE fed mice weighing significantly less than CON. FM increased over time in the control group but was unchanged in the KE group. Furthermore, LBM was not different between CON and KE mice despite KE mice weighing less than CON mice. Transcriptional analysis of skeletal muscle identified 6 genes that were significantly higher and 21 genes that were significantly lower in the KE group compared to CON. Lipidomic analysis of skeletal muscle identified no differences between groups for any lipid species, except for fatty acyl chains in triacylglycerol which was 46% lower in the KE group. Proteomics analysis identified 44 proteins that were different between groups, of which 11 were lower and 33 were higher in the KE group compared to CON. In conclusion, 72-week-old male mice consuming the exogenous KE, BD-AcAc2, had lower age-related gains in body weight and FM compared to CON mice. Furthermore, transcriptional and proteomics data suggest a signature in skeletal muscle of KE-treated mice consistent with markers of improved skeletal muscle regeneration, improved electron transport chain utilization, and increased insulin sensitivity.

α2,6 Sialylation mediated by ST6GAL1 promotes glioblastoma growth.

One of the least-investigated areas of brain pathology research is glycosylation, which is a critical regulator of cell surface protein structure and function. ß-Galactoside α2,6-sialyltransferase (ST6GAL1) is the primary enzyme that α2,6 sialylates N-glycosylated proteins destined for the plasma membrane or secretion, thereby modulating cell signaling and behavior. We demonstrate a potentially novel, protumorigenic role for α2,6 sialylation and ST6GAL1 in the deadly brain tumor glioblastoma (GBM). GBM cells with high α2,6 sialylation exhibited increased in vitro growth and self-renewal capacity and decreased mouse survival when orthotopically injected. α2,6 Sialylation was regulated by ST6GAL1 in GBM, and ST6GAL1 was elevated in brain tumor-initiating cells (BTICs). Knockdown of ST6GAL1 in BTICs decreased in vitro growth, self-renewal capacity, and tumorigenic potential. ST6GAL1 regulates levels of the known BTIC regulators PDGF Receptor ß (PDGFRB), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin, which were confirmed to bind to a lectin-recognizing α2,6 sialic acid. Loss of ST6GAL1 was confirmed to decrease PDGFRB α2,6 sialylation, total protein levels, and the induction of phosphorylation by PDGF-BB. Thus, ST6GAL1-mediated α2,6 sialylation of a select subset of cell surface receptors, including PDGFRB, increases GBM growth.

The SARS-CoV-2 Spike S1 Protein Induces Global Proteomic Changes in ATII-Like Rat L2 Cells that are Attenuated by Hyaluronan.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants that have been characterized to date, COVID-19 has led to 571,198,904 confirmed cases, and 6,387,863 deaths worldwide (as of July 15 th , 2022). Despite tremendous advances in our understanding of COVID19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics applications combined with systems biology analysis identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in an ATII-like Rat L2 cells that include three significant network hubs: E2F1, CREB1/ RelA, and ROCK2/ RhoA. Separately, we found that pre-treatment with High Molecular Weight Hyaluronan (HMW-HA), greatly attenuated the S1 effects. Immuno-targeted studies carried out on E2F1 and Rock2/ RhoA induction and kinase-mediated activation, in addition to cell cycle measurements, validated these observations. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with standard immuno-targeted and cell cycle measurements revealed profound and novel biological changes that contribute to our current understanding of both Spike S1 and Hyaluronan biology. This data shows that the Spike S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV2 induced cell injury.

Engineered colorectal cancer tissue recapitulates key attributes of a patient-derived xenograft tumor line.

The development of physiologically relevantin vitrocolorectal cancer (CRC) models is vital for advancing understanding of tumor biology. Although CRC patient-derived xenografts (PDXs) recapitulate key patient tumor characteristics and demonstrate high concordance with clinical outcomes, the use of thisin vivomodel is costly and low-throughput. Here we report the establishment and in-depth characterization of anin vitrotissue-engineered CRC model using PDX cells. To form the 3D engineered CRC-PDX (3D-eCRC-PDX) tissues, CRC PDX tumors were expandedin vivo, dissociated, and the isolated cells encapsulated within PEG-fibrinogen hydrogels. Following PEG-fibrinogen encapsulation, cells remain viable and proliferate within 3D-eCRC-PDX tissues. Tumor cell subpopulations, including human cancer and mouse stromal cells, are maintained in long-term culture (29 days); cellular subpopulations increase ratiometrically over time. The 3D-eCRC-PDX tissues mimic the mechanical stiffness of originating tumors. Extracellular matrix protein production by cells in the 3D-eCRC-PDX tissues resulted in approximately 57% of proteins observed in the CRC-PDX tumors also being present in the 3D-eCRC-PDX tissues on day 22. Furthermore, we show congruence in enriched gene ontology molecular functions and Hallmark gene sets in 3D-eCRC-PDX tissues and CRC-PDX tumors compared to normal colon tissue, while prognostic Kaplan-Meier plots for overall and relapse free survival did not reveal significant differences between CRC-PDX tumors and 3D-eCRC-PDX tissues. Our results demonstrate high batch-to-batch consistency and strong correlation between ourin vitrotissue-engineered PDX-CRC model and the originatingin vivoPDX tumors, providing a foundation for future studies of disease progression and tumorigenic mechanisms.

Integrated bioinformatics analysis identifies established and novel TGFß1-regulated genes modulated by anti-fibrotic drugs.

Fibrosis is a leading cause of morbidity and mortality worldwide. Although fibrosis may involve different organ systems, transforming growth factor-ß (TGFß) has been established as a master regulator of fibrosis across organs. Pirfenidone and Nintedanib are the only currently-approved drugs to treat fibrosis, specifically idiopathic pulmonary fibrosis, but their mechanisms of action remain poorly understood. To identify novel drug targets and uncover potential mechanisms by which these drugs attenuate fibrosis, we performed an integrative 'omics analysis of transcriptomic and proteomic responses to TGFß1-stimulated lung fibroblasts. Significant findings were annotated as associated with pirfenidone and nintedanib treatment in silico via Coremine. Integrative 'omics identified a co-expressed transcriptomic and proteomic module significantly correlated with TGFß1 treatment that was enriched (FDR-p = 0.04) with genes associated with pirfenidone and nintedanib treatment. While a subset of genes in this module have been implicated in fibrogenesis, several novel TGFß1 signaling targets were identified. Specifically, four genes (BASP1, HSD17B6, CDH11, and TNS1) have been associated with pirfenidone, while five genes (CLINT1, CADM1, MTDH, SYDE1, and MCTS1) have been associated with nintedanib, and MYDGF has been implicated with treatment using both drugs. Using the Clue Drug Repurposing Hub, succinic acid was highlighted as a metabolite regulated by the protein encoded by HSD17B6. This study provides new insights into the anti-fibrotic actions of pirfenidone and nintedanib and identifies novel targets for future mechanistic studies.

Local SARS-CoV-2 Peptide-Specific Immune Responses in Lungs of Convalescent and Uninfected Human Subjects.

Multi-specific and long-lasting T cell immunity have been recognized as indicators for long term protection against pathogens including the novel coronavirus SARS-CoV-2, the causative agent of the COVID-19 pandemic. Functional significance of peripheral memory T cells in individuals recovering from COVID-19 (COVID-19 + ) are beginning to be appreciated; but little is known about lung resident memory T cells (lung TRM) in SARS-CoV-2 infection. Here, we utilize a perfused three dimensional (3D) human lung tissue model and identify pre-existing local T cell immunity against SARS-CoV-2 proteins in lung tissues. We report ex vivo maintenance of functional multi-specific IFN-γ secreting lung TRM in COVID-19 + and their induction in lung tissues of vaccinated COVID-19 + . Importantly, we identify SARS-CoV-2 peptide-responding B cells and IgA + plasma cells in lung tissues of COVID-19 + in ex vivo 3D-tissue models. Our study highlights the importance of balanced and local anti-viral immune response in the lung with persistent induction of TRM and IgA + plasma cells for future protection against SARS-CoV-2 infection. Further, our data suggest that inclusion of multiple viral antigens in vaccine approaches may broaden the functional profile of memory T cells to combat the severity of coronavirus infection.

Molecular Tumor Board Review and Improved Overall Survival in Non-Small-Cell Lung Cancer.

With the introduction of precision medicine, treatment options for non-small-cell lung cancer have improved dramatically; however, underutilization, especially in disadvantaged patients, like those living in rural Appalachian regions, is associated with poorer survival. Molecular tumor boards (MTBs) represent a strategy to increase precision medicine use. UK HealthCare at the University of Kentucky (UK) implemented a statewide MTB in January 2017. We wanted to test the impact of UK MTB review on overall survival in Appalachian and other regions in Kentucky. METHODS: We performed a case-control study of Kentucky patients newly diagnosed with non-small-cell lung cancer between 2017 and 2019. Cases were reviewed by the UK MTB and were compared with controls without UK MTB review. Controls were identified from the Kentucky Cancer Registry and propensity-matched to cases. The primary end point was the association between MTB review and overall patient survival. RESULTS: Overall, 956 patients were included, with 343 (39%) residing in an Appalachian region. Seventy-seven (8.1%) were reviewed by the MTB and classified as cases. Cox regression analysis showed that poorer survival outcome was associated with lack of MTB review (hazard ratio [HR] = 8.61; 95% CI, 3.83 to 19.31; P < .0001) and living in an Appalachian region (hazard ratio = 1.43; 95% CI, 1.17 to 1.75; P = .004). Among individuals with MTB review, survival outcomes were similar regardless of whether they lived in Appalachia or other parts of Kentucky. CONCLUSION: MTB review is an independent positive predictor of overall survival regardless of residence location. MTBs may help overcome some health disparities for disadvantaged populations.

Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells.

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.

Many tissues in the body are capable of regenerating by replacing defective or worn-out cells with new ones. This process relies heavily on stem cells, which are precursor cells that lack a set role in the body and can develop into different types of cells under the right conditions. Tissues often have their own pool of stem cells that they use to replenish damaged cells. But as we age, this regeneration process becomes less effective. Many of our organs, such as the lungs, are lined with epithelial cells. These cells form a protective barrier, controlling what substances get in and out of the tissue. Alveoli are parts of the lungs that allow oxygen and carbon dioxide to move between the blood and the air in the lungs. And alveoli rely on an effective epithelial cell lining to work properly. To replenish these epithelial cells, alveoli have pockets, in which a type of epithelial cell, known as AEC2, lives. These cells can serve as stem cells, developing into a different type of cell under the right conditions. To work properly, AEC2 cells require close interactions with another type of cell called L-MSC, which supports the maintenance of other cells and also has the ability to differentiate into several other cell types. Both cell types can be found close together in these stem cell pockets. So far, it has been unclear how aging affects how these cells work together to replenish the epithelial lining of the alveoli. To investigate, Chanda et al. probed AEC2s and L-MSCs in the alveoli of young and old mice. The researchers collected both cell types from young (2-3 months) and aged (22-24 months) mice. Various combinations of these cells were grown to form 3D structures, mimicking how the cells grow in the lungs. Young L-MSCs formed normal 3D structures with both young and aged AEC2 cells. But aged L-MSCs developed abnormal, loose structures with AEC2 cells (both young and old cells). Aged L-MSCs were found to have higher levels of an enzyme (called Nox4) that produces oxidants and other 'pro-aging' factors, compared to young L-MSCs. However, reducing Nox4 levels in aged L-MSCs allowed these cells to form normal 3D structures with young AEC2 cells, but not aged AEC2 cells. These findings highlight the varying effects specific stem cells have, and how their behaviour is affected by pro-aging factors. Moreover, the pro-aging enzyme Nox4 shows potential as a therapeutic target ­ downregulating its activity may reverse critical effects of aging in cells.

Biomarkers for Adverse Lung Injury Following Pediatric Cardiopulmonary Bypass.

Cardiopulmonary bypass triggers systemic inflammation, resulting in lung injury, and frequently leads to prolonged mechanical ventilation. Biomarkers of systemic inflammation are required to predict the risk of such complications. We hypothesize that specific serum proteins can be used as biomarkers to predict the severity of lung injury following cardiac surgery. DESIGN: Retrospective chart review study. SETTING: Clinical variables were collected and used in conjuncture with unbiased proteomic analysis using mass spectrometry that was performed on frozen plasma samples from a study group (patients with mechanical ventilation > 48 hr post surgery) and a control group (patients with mechanical ventilation < 48 hr post surgery). SUBJECTS: Subjects included were infants who underwent cardiac surgery with similar complexity (Society of Thoracic Surgeons-European Association for Cardiothoracic Surgery 3 or 4) using cardiopulmonary bypass. Patients in both groups were matched for their weight, age, and duration of cardiopulmonary bypass. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Four-hundred eighty-three proteins were identified (99% minimum confidence and two peptides minimum, protein false discovery rate 0.1%) on proteomic analysis of four control and four study patients at precardiopulmonary bypass, 0, and 48 hours postcardiopulmonary bypass samples. Thirty-six of 178 proteins were significantly different (≥ 1.5-fold; p < 0.05) at precardiopulmonary bypass (top increased: tenascin; top decreased: tetranectin), 18 of 140 proteins at 0 hour (top increased: hemoglobin beta; top decreased: C8 beta), and 25 of 166 proteins at 48 hours post surgery (top increased: proteoglycan 4; top decreased: galectin-3-binding protein). The top pathway involved cytoskeleton remodeling. Other pathways involved immune response and blood coagulation. Proteoglycan 4 was validated by enzyme-linked immunosorbent assay in a different set of samples (n = 20/group; mean ± sd: 128 ± 67 vs 195 ± 160 ng/mL) (p = 0.037). CONCLUSIONS: Multiple proteomic biomarkers were associated with worse respiratory outcomes. Precardiopulmonary bypass biomarkers might indicate risk factors (e.g., abnormalities of coagulation), whereas those identified at 0 hour and post cardiopulmonary bypass may reflect mechanisms of ongoing pathobiology.