Pneumonia causes short‐ and long‐term cognitive dysfunction in a high proportion of patients, although the mechanism(s) responsible for this effect are unknown. Here, we tested the hypothesis that pneumonia‐elicited cytotoxic amyloid and tau variants: (1) are present in the circulation during infection; (2) lead to impairment of long‐term potentiation; and, (3) inhibit long‐term potentiation dependent upon tau. Cytotoxic amyloid and tau species were recovered from the blood and the hippocampus following pneumonia, and they were present in the extracorporeal membrane oxygenation oxygenators of patients with pneumonia, especially in those who died. Introduction of immunopurified blood‐borne amyloid and tau into either the airways or the blood of uninfected animals acutely and chronically impaired hippocampal information processing. In contrast, the infection did not impair long‐term potentiation in tau knockout mice and the amyloid‐ and tau‐dependent disruption in hippocampal signaling was less severe in tau knockout mice. Moreover, the infection did not elicit cytotoxic amyloid and tau variants in tau knockout mice. Therefore, pneumonia initiates a tauopathy that contributes to cognitive dysfunction.

Carbonic anhydrase IX (CA IX) is highly expressed in rapidly proliferating and highly glycolytic cells, where it serves to enhance acid-regulatory capacity. Pulmonary microvascular endothelial cells (PMVECs) actively utilize aerobic glycolysis and acidify media, whereas pulmonary arterial endothelial cells (PAECs) primarily rely on oxidative phosphorylation and minimally change media pH. Therefore, we hypothesized that CA IX is critical to PMVEC angiogenesis because of its important role in regulating pH. To test this hypothesis, PMVECs and PAECs were isolated from Sprague-Dawley rats. CA IX knockout PMVECs were generated using the CRISPR-Cas9 technique. During serum-stimulated growth, mild acidosis (pH 6.8) did not affect cell counts of PMVECs, but it decreased PAEC cell number. Severe acidosis (pH 6.2) decreased cell counts of PMVECs and elicited an even more pronounced reduction of PAECs. PMVECs had a higher CA IX expression compared with PAECs. CA activity was higher in PMVECs compared with PAECs, and enzyme activity was dependent on the type IX isoform. Pharmacological inhibition and genetic ablation of CA IX caused profound dysregulation of extra- and intracellular pH in PMVECs. Matrigel assays revealed impaired angiogenesis of CA IX knockout PMVECs in acidosis. Lastly, pharmacological CA IX inhibition caused profound cell death in PMVECs, whereas genetic CA IX ablation had little effect on PMVEC cell death in acidosis. Thus CA IX controls PMVEC pH necessary for angiogenesis during acidosis. CA IX may contribute to lung vascular repair during acute lung injury that is accompanied by acidosis within the microenvironment.

Background Acute lung injury (ALI) is a significant source of morbidity and mortality in critically ill patients. Age is a major determinant of clinical outcome in ALI. The increased ALI-associated mortality in the older population suggests that there are age-dependent alterations in the responses to pulmonary challenge. The objective of this observational study was to evaluate age-dependent differences in the acute (within 6hrs) immunological and physiological responses of the heart and lung, to pulmonary challenge, that could result in increased severity. Methods Male C57Bl/6 mice (young: 2–3 months, old: 18–20 months) were challenged intratracheally with cell wall components from gram positive bacteria (lipoteichoic acid and peptidoglycan). After 6h, both biochemical and physiological consequences of the challenge were assessed. Alveolar infiltration of inflammatory cells and protein, airspace and blood cytokines, cardiac function and myocardial proteasome activity were determined. Results In young mice there was a dose dependent response to pulmonary challenge resulting in increased airspace neutrophil counts, lung permeability, and concentrations of cytokines in bronchoalveolar lavage fluid and plasma. A midrange dose was then selected to compare the responses in young and old animals. In comparison, the old animals displayed increased neutrophil accumulation in the airspaces, decreased arterial oxygen saturation, body temperatures, plasma cytokine concentrations, and a lack of myocardial proteasome response, following challenge. Conclusions Age dependent differences in the onset of systemic response and in maintenance of vital functions, including temperature control, oxygen saturation and myocardial proteasome activation, are evident. We believe a better understanding of these age-related consequences of ALI can lead to more appropriate treatments in the elderly patient population.

Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid that they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared, including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 h with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-d-glucose had no effect on migration but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.

Pulmonary artery, capillary, and vein endothelial cells possess distinctive structures and functions, which represent a form of vascular segment specific macroheterogeneity. However, within each of these segmental populations, individual cell functional variability represents a poorly characterized microheterogeneity. Here, we hypothesized that single cell clonogenic assays would reveal microheterogeneity among the parent cell population and enable isolation of highly representative cells with committed parental characteristics. To test this hypothesis, pulmonary microvascular endothelial cells (PMVECs) and pulmonary arterial endothelial cells (PAECs) were isolated from different Sprague Dawley rats. Serum stimulated proliferation of endothelial populations and single cell clonogenic potential were evaluated. In vitro Matrigel assays were utilized to analyze angiogenic potential and the Seahorse assay was used to evaluate bioenergetic profiles. PMVEC populations grew faster and had a higher proliferative potential than PAEC populations. Fewer PMVECs were needed to form networks on Matrigel when compared with PAECs. PMVECs primarily utilized aerobic glycolysis, while PAECs relied more heavily on oxidative phosphorylation, to support bioenergetic demands. Repeated single cell cloning and expansion of PAEC colonies generated homogeneous first-generation clones that were highly reflective of the parental population in terms of growth, angiogenic potential, and bioenergetic profiles. Repeated single cell cloning of the first-generation clones generated second-generation clones with increased proliferative potential while maintaining other parental characteristics. Second-generation clones were highly homogeneous populations. Thus, single cell cloning reveals microheterogeneity among the parent cell population and enables isolation of highly representative cells with parental characteristics.

Pulmonary hypertension is a complex, multifactorial disease that results in right heart failure and premature death. Since the initial reports of pulmonary hypertension in the late 1800s, the diagnosis of pulmonary hypertension has evolved with respect to its definition, screening tools, and diagnostic techniques. This historical perspective traces the earliest roots of pulmonary hypertension detection and diagnosis through to the current recommendations for classification. We highlight the diagnostic tools used in the past and present, and end with a focus on the future directions of early detection. Early detection of pulmonary hypertension and pulmonary arterial hypertension and the proper determination of etiology are vital for the early therapeutic intervention that can prolong life expectancy and improve quality of life. The search for a non-invasive screening tool for the identification and classification of pulmonary hypertension is ongoing, and we discuss the role of animal models of the disease in this search.

The aging lung is increasingly susceptible to infectious disease. Changes in pulmonary physiology and function are common in older populations, and in those older than 60 years, pneumonia is the major cause of infectious death. Understanding age-related changes in the innate and adaptive immune systems, and how they affect both pulmonary and systemic responses to pulmonary challenge are critical to the development of novel therapeutic strategies for the treatment of the elderly patient. In this observational study, we examined age-associated differences in inflammatory responses to pulmonary challenge with cell wall components from Gram-positive bacteria. Thus, male Sprague–Dawley rats, aged 6 months or greater than 18 months (approximating humans of 20 and 55–65 years), were challenged, intratracheally, with lipoteichoic acid and peptidoglycan. Cellular and cytokine evaluations were performed on both bronchoalveolar lavage fluid (BAL) and plasma, 24 h post-challenge. The plasma concentration of free thyroxine, a marker of severity in non-thyroidal illness, was also evaluated. The older animals had an increased chemotactic gradient in favor of the airspaces, which was associated with a greater accumulation of neutrophils and protein. Furthermore, macrophage migration inhibitory factor (MIF), an inflammatory mediator and putative biomarker in acute lung injury, was increased in both the plasma and BAL of the older, but not young animals. Conversely, plasma free thyroxine, a natural inhibitor of MIF, was decreased in the older animals. These findings identify age-associated inflammatory/metabolic changes following pulmonary challenge that it may be possible to manipulate to improve outcome in the older, critically ill patient.

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.