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Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)-induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS cohort and correlated with increased plasma angiopoietin 2 levels. COVID-19 ARDS lung autopsy immunostaining confirmed a link between vascular injury (angiopoietin 2) and platelet-rich microthrombi (CD61) and induction of necrotic cell death [phosphorylated mixed lineage kinase domain-like (pMLKL)]. Among recovery subjects, the vascular signature identified patients with poor functional outcomes. Taken together, this vascular injury signature was associated with low platelet levels and increased mortality and can be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated lung lymphatic function in the pathogenesis of CS-induced emphysema. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that preceded emphysema. Proteomic analysis of lymph confirmed upregulation of coagulation and inflammatory pathways in the lymphatics of CS-exposed mice compared to control mice. These data suggest that CS exposure results in lung lymphatic dysfunction with thrombosis, impaired leukocyte trafficking, and changes in the composition of lymph. In patients with emphysema, lung lymphatic thrombosis is seen with increasing disease severity. These studies for the first time demonstrate lung lymphatic dysfunction after cigarette smoke exposure and suggest a novel component in the pathogenesis of emphysema.

The novel coronavirus disease-19 (COVID-19) pandemic caused by SARS-CoV-2 has ravaged global healthcare with previously unseen levels of morbidity and mortality. To date, methods to predict the clinical course, which ranges from the asymptomatic carrier to the critically ill patient in devastating multi-system organ failure, have yet to be identified. In this study, we performed large-scale integrative multi-omics analyses of serum obtained from COVID-19 patients with the goal of uncovering novel pathogenic complexities of this disease and identifying molecular signatures that predict clinical outcomes. We assembled a novel network of protein-metabolite interactions in COVID-19 patients through targeted metabolomic and proteomic profiling of serum samples in 330 COVID-19 patients compared to 97 non-COVID, hospitalized controls. Our network identified distinct protein-metabolite cross talk related to immune modulation, energy and nucleotide metabolism, vascular homeostasis, and collagen catabolism. Additionally, our data linked multiple proteins and metabolites to clinical indices associated with long-term mortality and morbidity, such as acute kidney injury. Finally, we developed a novel composite outcome measure for COVID-19 disease severity and created a clinical prediction model using a set of 33 metabolites. The model significantly improved the identification of key events of critical illness such as prolonged hospitalization, supplemental oxygen requirement, acute kidney injury (AKI), and acute respiratory distress syndrome (ARDS), beyond those achieved using the more traditional risk factors of age, gender, and BMI.

Vascular injury is a menacing element of acute respiratory distress syndrome (ARDS) pathogenesis. To better understand the role of vascular injury in COVID-19 ARDS, we used lung autopsy immunohistochemistry and blood proteomics from COVID-19 subjects at distinct timepoints in disease pathogenesis, including a hospitalized cohort at risk of ARDS development (“at risk”, N=59), an intensive care unit cohort with ARDS (“ARDS”, N=31), and a cohort recovering from ARDS (“recovery”, N=12). COVID-19 ARDS lung autopsy tissue revealed an association between vascular injury and platelet-rich microthrombi. This link guided the derivation of a protein signature in the at risk cohort characterized by lower expression of vascular proteins in subjects who died, an early signal of vascular limitation termed the maladaptive vascular response. These findings were replicated in COVID-19 ARDS subjects, as well as when bacterial and influenza ARDS patients (N=29) were considered, hinting at a common final pathway of vascular injury that is more disease (ARDS) then cause (COVID-19) specific, and may be related to vascular cell death. Among recovery subjects, our vascular signature identified patients with good functional recovery one year later. This vascular injury signature could be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

COVID-19 outcomes like mortality have been associated with albumin alteration. However, it is unclear whether albumin changes in COVID-19 are pathogen specific or not. To this end, we characterized the kinetics of serum albumin in mechanically ventilated patients with COVID-19 compared to mechanically ventilated patients with sepsis-induced Acute Respiratory Distress Syndrome (ARDS). We discovered two phases of alterations in albumin levels during the course of Covid-19 critical illness, but not for the sepsis-induced ARDS. Our findings suggest the metabolic effects of COVID-19 are pathogen-specific and albumin recovery may signal the cessation of a deleterious immune response in this disease.

Respiratory failure and acute kidney injury (AKI) are associated with high mortality in SARS-CoV-2-associated Coronavirus disease 2019 (COVID-19). These manifestations are linked to a hypercoaguable, pro-inflammatory state with persistent, systemic complement activation. Three critical COVID-19 patients recalcitrant to multiple interventions had skin biopsies documenting deposition of the terminal complement component C5b-9, the lectin complement pathway enzyme MASP2, and C4d in microvascular endothelium. Administration of anti-C5 monoclonal antibody eculizumab led to a marked decline in D-dimers and neutrophil counts in all three cases, and normalization of liver functions and creatinine in two. One patient with severe heart failure and AKI had a complete remission. The other two individuals had partial remissions, one with resolution of his AKI but ultimately succumbing to respiratory failure, and another with a significant decline in FiO2 requirements, but persistent renal failure. In conclusion, anti-complement therapy may be beneficial in at least some patients with critical COVID-19., Highlights • SARS-CoV-2 infection in COVID-19 is associated with sustained complement activation. • Severe COVID-19 involves a pro-inflammatory/hypercoaguable state linked to complement. • Complement deposition in skin of 3 COVID-19 cases recalcitrant to therapy is shown. • Anti-C5 therapy with eculizumab led to 1 complete and 2 partial clinical remissions. • Our data may inform guidelines for earlier intervention with anti-C agents in COVID-19.

The development of chronic lung disease occurs as a consequence of multiple cellular events that involve an initial insult which often leads to the development of chronic inflammation, and the dysregulation of cellular proliferation and cell death mechanisms. Multiple cell types in the lung are key to the respiratory and protective/barrier functions necessary to manage the chronic exposures to environmental, mechanical, and oxidative stressors. Autophagy is essential to lung development and homeostasis, as well as the prevention and development of disease. The cellular process involves the collection and removal of unwanted organelles and proteins through lysosomal degradation. In recent years, investigations have addressed the roles of autophagy and selective autophagy in numerous chronic lung diseases. Here, we highlight recent advances on the role of autophagy in the pathogenesis of asthma, chronic obstructive pulmonary disease and emphysema, pulmonary arterial hypertension, and idiopathic pulmonary fibrosis.

Background: Chronic obstructive pulmonary disease (COPD) is a common disease characterized by persistent respiratory symptoms and airflow restriction. The mechanisms underlying pathogenesis in COPD are still poorly understood. Identification of the dysfunctional genes in human lung from patients with Chronic obstructive pulmonary disease (COPD) will help up to understand the pathology of this disease. To identify the dysfunctional genes in human lung from patients with Chronic obstructive pulmonary disease (COPD). We used transcriptomic data of lung tissue for 91 COPD cases and 182 matched healthy controls from the Genotype-Tissue Expression (GTEx) database. We employed a stringent model controlling for known covariates and hidden confounders. DESeq2 R package (v1.20.0) was used to test for differential expression.Results: We identified 1,359 significant differentially expressed genes (DEG) with 707 upregulated and 602 downregulated respectively. We evaluated the identified DEGs in an independent microarray cohort of 219 COPD and 108 controls, demonstrating the robustness of our result. Functional annotation of COPD-associated genes highlighted the activation of complement cascade, dysregulation of inflammatory response and extracellular matrix organization in the COPD patients. In addition, we identified several novel key-hub genes involved in the COPD pathogenesis using a network analysis method.Conclusion: In summary, our study represents the comprehensive analysis of gene expression on COPD with the largest sample size, providing great resource for the molecular research in the COPD community.

Rationale: Although the transplantation of induced pluripotent stem cell (iPSC)–derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC–derived cells in lung disease models are missing. Objectives: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte–macrophage colony–stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. Methods: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. Measurements and Main Results: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. Conclusions: We here demonstrate for the first time that pulmonary transplantation of human iPSC–derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

Persistent inflammation within the respiratory tract underlies the pathogenesis of numerous chronic pulmonary diseases including chronic obstructive pulmonary disease, asthma and pulmonary fibrosis. Chronic inflammation in the lung may arise from a combination of genetic susceptibility and environmental influences, including exposure to microbes, particles from the atmosphere, irritants, pollutants, allergens, and toxic molecules. To this end, an immediate, strong, and highly regulated inflammatory defense mechanism is needed for the successful maintenance of homeostasis within the respiratory system. Macroautophagy/autophagy plays an essential role in the inflammatory response of the lung to infection and stress. At baseline, autophagy may be critical for inhibiting spontaneous pulmonary inflammation and fundamental for the response of pulmonary leukocytes to infection; however, when not regulated, persistent or inefficient autophagy may be detrimental to lung epithelial cells, promoting lung injury. This perspective will discuss the role of autophagy in driving and regulating inflammatory responses of the lung in chronic lung diseases with a focus on potential avenues for therapeutic targeting. Abbreviations AR allergic rhinitis AM alveolar macrophage ATG autophagy-related CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator COPD chronic obstructive pulmonary disease CS cigarette smoke CSE cigarette smoke extract DC dendritic cell IH intermittent hypoxia IPF idiopathic pulmonary fibrosis ILD interstitial lung disease MAP1LC3B microtubule associated protein 1 light chain 3 beta MTB Mycobacterium tuberculosis MTOR mechanistic target of rapamycin kinase NET neutrophil extracellular traps OSA obstructive sleep apnea PAH pulmonary arterial hypertension PH pulmonary hypertension ROS reactive oxygen species TGFB1 transforming growth factor beta 1 TNF tumor necrosis factor.

Pemetrexed represents the first antifolate cancer drug to be approved by the Food and Drug Administration in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non–small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis that becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP. [Cancer Res 2009;69(13):5467–74]

As antimicrobial resistance increases, understanding the current epidemiology of bloodstream infections (BSIs) in hematopoietic stem cell transplant (HSCT) recipients is essential to guide empirical antimicrobial therapy. We therefore reviewed microbial etiologies, timing and outcomes of BSIs in patients who were transplanted from September 2007 to December 2011. Vancomycin-resistant enterococci (VRE) were the most common pathogens in allogeneic HSCT recipients and the fourth most common after autologous transplant. VRE did not cause any of 101 BSIs in neutropenic patients who were not receiving antibacterials, but caused 32 (55%) of 58 BSIs in neutropenic patients receiving a broad-spectrum β-lactam agent (p < 0.001). Rates of septic shock and 7-day mortality were 5% and 0% for streptococcal bacteremia, 12% and 18% for VRE bacteremia, and 20% and 14% for Gram-negative bacteremia. In conclusion, VRE bacteremia was the most common BSI in allogeneic HSCT recipients, occurred primarily in neutropenic patients receiving broad-spectrum β-lactams and was associated with poor outcomes.

The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMP-activated kinase (AMPK). Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. Whereas AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1-dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs. Cancer Res; 70(24); 10299–309. ©2010 AACR.

The mouse fpgs gene uses two distantly placed promoters to produce functionally distinct isozymes in a tissue-specific pattern. We queried how the P1 and P2 promoters were differentially controlled. DNA methylation of the CpG-sparse P1 promoter occurred only in tissues not initiating transcription at this site. The P2 promoter, which was embedded in a CpG island, appeared open to transcription in all tissues by several criteria, including lack of DNA methylation, yet was used only in dividing tissues. The patterns of histone modifications over the two promoters were very different: over P1, histone activation marks (acetylated histones H3 and H4 and H3 trimethylated at K4) reflected transcriptional activity and apparently reinforced the effects of hypomethylated CpGs; over P2, these marks were present in tissues whether P2 was active, inactive, or engaged in assembly of futile initiation complexes. Since P1 transcriptional activity coexisted with silencing of P2, we sought the mechanism of this transcriptional interference. We found RNA polymerase II, phosphorylated in a pattern consistent with transcriptional elongation, and only minimal levels of initiation factors over P2 in liver. We concluded that mouse fpgs uses DNA methylation to control tissue-specific expression from a CpG-sparse promoter, which is dominant over a downstream promoter masked by promoter occlusion.

AMP-activated protein kinase (AMPK) is a major cellular energy sensor, functioning as a regulator of signaling cascades controlling protein, lipid, and carbohydrate metabolism. Because of the dependence of many tumors on the deregulation of the PI3K-AKT-mTOR signaling cascade, the discovery of AMPK activators is an attractive approach for cancer therapeutics, aimed at reinstating control over cellular metabolic functions altered during carcinogenesis rather than inhibiting cellular targets. The antifolate pemetrexed is FDA approved for first line treatment of mesothelioma and non-small cell lung cancer (NSCLC). While pemetrexed acts primarily as a thymidylate synthase inhibitor, we discovered that the drug is also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. The inhibition of AICART by pemetrexed resulted in a marked accumulation of the reaction substrate, ZMP, a known allosteric activator of AMPK in several human carcinoma cell lines. The mammalian target of rapamycin complex 1 (mTORC1) was inhibited following pemetrexed treatment, as determined by hypophosphorylation of S6K1 and 4EBP1, and this effect was dependent on AMPK activation. Co-administration of GSK690693, a pan AKT inhibitor prolonged mTORC1 inhibition following pemetrexed treatment and offset the consequences of negative feedback activation of mTORC1 through PI3-kinase and mTORC2 activity. Interestingly, cells insensitive to mTORC1 inhibition following AMPK activation retained sensitivity to the secondary effect of pemetrexed, apparently due to inhibition of lipid synthesis through phosphorylation of acetyl-CoA carboxylase by AMPK. We concluded that the growth inhibitory effect of pemetrexed following AMPK activation in human carcinoma cells was due to a combined effect of inhibition of mTORC1 signaling and lipogenesis, both of which were mediated by activation of AMPK. Somewhat surprisingly, several cell lines null for LKB1, the major upstream kinase of AMPK, retained sensitivity to the secondary effects of pemetrexed mediated through AMPK activation. This suggests that the effect of pemetrexed on AMPK activity will be preserved in tumors lacking LKB1, a common genotype of human NSCLC. Hence, pemetrexed exerts its effects on carcinoma cells by both inhibition of traditional antifolate targets and by a novel mechanism, activation of AMPK. (Supported in part by NIH Grant CA-27605) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5086.