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BackgroundPulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs.MethodsLung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1.ResultsIn lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs.ConclusionVascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

Objective There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Methods Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 hours and 10 days after transplantation and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using anti-human CD63 antibody quantum dot on the nanoparticle detector, and via GFP trigger on the nanoparticle flow cytometer (FACS). Results In syngeneic controls, steady state levels of circulating donor exosomes were detected at all post-transplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. Conclusions Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

Circadian rhythms are endogenous oscillations that occur with a 24-hr periodicity. These rhythms are ubiquitous and thus, vascular endothelial cells that line the vascular bed are also subjected to circadian regulation. While the circadian control of vascular function has been demonstrated in the context of various pathologies, the relevance and functional implication of clock control over pulmonary vasculature has never been investigated. As the pulmonary endothelium is a crucial site for the host’s inflammatory response to a lung specific pathogen, we investigated the role of the circadian clock in mediation the response of the pulmonary endothelium to inflammation. We hypothesized that the pulmonary endothelium is under circadian control and that the clock serves to curb inflammatory signaling.MethodsCircadian rhythms were monitored in pulmonary artery segments and endothelial cells isolated from mPer2luciferase transgenic mice in the presence of an inflammatory stimuli (LPS). Reactive oxygen species (ROS) production in LPS treated cells was measured by fluorescence microscopy using the cell permeant dye CellROX Green. NLRP3 inflammasome was monitored post-mortem (0-72 h post LPS instillation) by measuring the expression of the NLRP3 subunit in wild type and Bmal1−/− and Cry1/2−/− mice. Inflammation was quantified in these mice by measuring PMN adherence and intercellular adhesion molecule (ICAM-1).ResultsWe observed that the circadian rhythm of the pulmonary vasculature was altered LPS. LPS also led to ROS production in these cells; ROS increased 3 h post LPS treatment, peaked by 36 h and returned to baseline values by 72 h. ROS were inhibited by pretreating the cells with the NADPH oxidase 2 (NOX2) inhibitor dipheneylene iodonium (DPI). Addition of DPI, prior to LPS pretreatment also restored the circadian rhythmicity of the pulmonary endothelium. The increase in NLRP3 along the vessel wall (post LPS treatment) was resolved by 72 h in lungs of wild type mice but not in Bmal1−/− and Cry1−/−Cry2−/− lungs. Inflammation (ICAM-1 and PMN) was also resolved in wild type but not in mice wherein the circadian clock had been disrupted genetically.ConclusionOur data indicate that pro-inflammatory stimuli reprogram circadian rhythms in the pulmonary endothelium via ROS via the NOX2-NLRP3 pathway. Disruption of the clock mediates a sustained increase in ROS via this Nox2-NLRP3 pathway in endothelial cells, thus offering a novel mechanism for mitigating the effects of clock disruption.

The effects of e-cigarette (e-cig) aerosol inhalation by nonsmokers have not been examined to date. The present study was designed to evaluate the acute response to aerosol inhalation of non-nicotinized e-cigarettes in terms of oxidative stress and indices of endothelial activation in human pulmonary microvascular endothelial cells (HPMVEC). Ten smoking-naïve healthy subjects (mean age ± SD = 28.7 ± 5.5 yr) were subjected to an e-cig challenge, following which their serum was monitored for markers of inflammation [C-reactive protein (CRP) and soluble intercellular adhesion molecule (sICAM)] and nitric oxide metabolites (NOx). The oxidative stress and inflammation burden of the circulating serum on the vascular network was also assessed by measuring reactive oxygen species (ROS) production and induction of ICAM-1 expression on HPMVEC. Our results show that serum indices of oxidative stress and inflammation increased significantly ( P < 0.05 as compared with baseline), reaching a peak at approximately 1–2 h post-e-cig aerosol inhalation and returning to baseline levels at 6 h. The circulatory burden of the serum (ICAM-1 and ROS) increased significantly at 2 h and returned to baseline values 6 h post-e-cig challenge. ROS production by HPMVEC was found to occur via activation of the NADPH oxidase 2 (NOX2) pathways. These findings suggest that even in the absence of nicotine, acute e-cig aerosol inhalation leads to a transient increase in oxidative stress and inflammation. This can adversely affect the vascular endothelial network by promoting oxidative stress and immune cell adhesion. Thus e-cig inhalation has the potential to drive the onset of vascular pathologies.

Pulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs.Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were HE stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1.In lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs.Vascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

Hyperinflammation is a key event that occurs with SARS-CoV-2 infection. In the lung, hyperinflammation leads to structural damage to tissue. To date, numerous lung histological studies have shown extensive alveolar damage, but there is scarce documentation of vascular inflammation in postmortem lung tissue. Here we document histopathological features and monitor the NLRP3 inflammasome in fatal cases of disease caused by SARS Cov2 (COVID-19). We posit that inflammasome formation along the vessel wall is a characteristic of lung inflammation that accompanies COVID-19 and that it is a probable candidate that drives amplification of inflammation post infection.

The vascular system is a network that consists of a pump (the heart) that provides the force for flow of blood through arteries, capillaries, and veins. This network transports oxygen and nutrients to cells and removes the wastes. The circulatory system also includes the lymphatic system via which “leaked” fluid from the interstitial space of tissues is brought back to the venous system. The vascular network is crucial in maintenance of life and its dysfunction induces pathologies including cardiovascular diseases (e.g., hypertension, atherosclerosis, and restenosis), tumor angiogenesis, and cancer metastasis. The endothelium that forms the inner lining of the vascular network plays a key role in regulating the function of this network. This chapter will review the current knowledge of the endothelial structure and function and the myriad endothelial signaling pathways that regulate processes ranging from inflammation and angiogenesis to oxidative stress and immune function.

This study was designed to investigate the acute effects of nonnicotinized e-cigarette (e-cig) aerosol inhalation in nonsmokers both in terms of blood-based markers of inflammation and oxidative stress and evaluate their association with hemodynamic-metabolic MRI parameters quantifying peripheral vascular reactivity, cerebrovascular reactivity, and aortic stiffness. Thirty-one healthy nonsmokers were subjected to two blood draws and two identical MRI protocols, each one before and after a standardized e-cig vaping session. After vaping, the serum levels of C-reactive protein, soluble intercellular adhesion molecule, and the danger signal machinery high-mobility group box 1 (HMGB1) and its downstream effector and the NLR family pyrin domain containing 3 (NLRP3) inflammasome (as monitored by its adaptor protein ASC) increased significantly relative to the respective baseline (prevaping) values. Moreover, nitric oxide metabolites and reactive oxygen species production decreased and increased, respectively. These observations were paralleled by impaired peripheral vascular reactivity (with reduced flow-mediated dilation and attenuated hyperemic response after a cuff-occlusion test) and metabolic alterations expressed by decreased venous oxygen saturation, postvaping. The current results suggest propagation of inflammation signaling via activation of the danger signaling axis (HMGB1-NLRP3). The findings indicate that a single episode of vaping has adverse impacts on vascular inflammation and function. NEW & NOTWORTHY Endothelial cell signaling and blood biomarkers were found to correlate with functional vascular changes in a single episode e-cigarettes inhalation in healthy adults. This is indicative of the potential of e-cigarettes (even when inhaled acutely) to lead of vascular dysfunction.

Introduction: While the circadian clock has been characterized in systemic blood vessels and well established to contribute to various inflammatory pathologies associated with the endothelium, the mechanisms of circadian control of endothelial inflammation specifically in pulmonary inflammation is not known. We hypothesized that the pulmonary vasculature is under circadian control and that this rhythm is disrupted by inflammatory stimuli via redox mediated processes. Objective:To determine the possible relationship between the inflammation induced disruption in Circadian Rhythm in pulmonary endothelial cells and reactive oxygen species. Material and Methods: Circadian rhythms were monitored in pulmonary artery segments and endothelial cells isolated from mPer2luciferase transgenic mice in presence of an inflammatory stimuli (LPS). Reactive oxygen species (ROS) production in LPS treated cells was measured by fluorescence microscopy using the cell permeant dye CellROX Green. Results: The circadian rhythm of the pulmonary endothelium was disrupted by LPS. To identify the mechanism of this disruption, ROS production in these cells was monitored. At 3 h post LPS treatment, we observed a >3 fold increase in ROS production which further increased to 6 fold by 36 h and returned to baseline values at 72 h. ROS was inhibited by pretreating the cells with the NADPH oxidase 2 (NOX2) inhibitor DPI. Addition of DPI, prior to LPS pretreatment also restored the circadian rhythmicity of the pulmonary endothelium. Conclusions: Pro-inflammatory stimuli can disrupt circadian rhythms in the pulmonary endothelium via NOX2 regulated ROS signaling. We speculate that under inflammatory conditions disrupted circadian rhythms in the pulmonary vascular endothelium contributes to worsening outcomes.

Updated measurements of charged particle fluxes during the transit from Earth to Mars as well as on site measurements by Curiosity of Martian surface radiation fluxes identified potential health hazards associated with radiation exposure for human space missions. Designing mitigation strategies of radiation risks to astronauts is critical. We investigated radiation-induced endothelial cell damage and its mitigation by LGM2605, a radioprotector with antioxidant and free radical scavenging properties. We used an in vitro model of lung vascular networks (flow-adapted endothelial cells, FAECs), exposed to gamma rays, low/higher linear energy transfer (LET) protons (3&ndash, 4 or 8&ndash, 10 keV/&micro, m, respectively), and mixed field radiation sources (gamma and protons), given at mission-relevant doses (0.25 gray (Gy)&ndash, 1 Gy). We evaluated endothelial inflammatory phenotype, NLRP3 inflammasome activation, and oxidative cell injury. LGM2605 (100 &micro, M) was added 30 min post radiation exposure and gene expression changes evaluated 24 h later. Radiation induced a robust increase in mRNA levels of antioxidant enzymes post 0.25 Gy and 0.5 Gy gamma radiation, which was significantly decreased by LGM2605. Intercellular cell adhesion molecule-1 (ICAM-1) and NOD-like receptor protein 3 (NLRP3) induction by individual or mixed-field exposures were also significantly blunted by LGM2605. We conclude that LGM2605 is a likely candidate to reduce tissue damage from space-relevant radiation exposure.

The current standard for non-invasive imaging of acute rejection consists of X-ray/CT, which derive their contrast from changes in ventilation, inflammation and edema, as well as remodeling during rejection. We propose the use of hyperpolarized [1-(13)C] pyruvate MRI—which provides real-time metabolic assessment of tissue—as an early biomarker for tissue rejection. In this preliminary study, we used μCT-derived parameters and HP (13)C MR-derived biomarkers to predict rejection in an orthotopic left lung transplant model in both allogeneic and syngeneic rats. On day 3, the normalized lung density—a parameter that accounts for both lung volume (mL) and density (HU)—was −0.335 (CI:−0.598,−0.073) and −0.473 (CI:−0.726,−0.220) for the allograft and isograft, respectively (NS, p=0.40). The lactate-to-pyruvate ratios—derived from the HP (13)C MRI— for the allograft and isograft were 0.200 (CI:0.161,0.240) and 0.114 (CI:0.074,0.153), respectively (significant, p=0.020). Both techniques show tissue rejection on day 7. A separate sub-study revealed CD8+ cells as the primary source of the lactate-to-pyruvate signal. Our study suggests that hyperpolarized (HP) [1-(13)C] pyruvate MRI is a promising early biomarker for tissue rejection that provides metabolic assessment in real-time based on changes in cellularity and metabolism of lung tissue and the infiltrating inflammatory cells, and may be able to predict tissue rejection earlier than X-ray/CT.

Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A2 and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H2O2 and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O2). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H2O2 or short chain hydroperoxides does not play a role in the recovery process., Graphical abstract Conclusion: all 3 enzymatic activities of Prdx6 contribute to the reversal of cell membrane phospholipid peroxidation.fx1, Highlights • Repair of peroxidized lipids did not occur with H26A-Prdx6 Delete semicolons;mutation. • Repair reflects the phospholipid hydroperoxidase and PLA2 activities of Prdx6;Move to next with "bullet mark" "P"eroxidase activity with small hydroperoxides and H2O2 does not play a role in repair.

The current standard for noninvasive imaging of acute rejection consists of X-ray/CT, which derive their contrast from changes in ventilation, inflammation and edema, as well as remodeling during rejection. We propose the use of hyperpolarized [1-13 C] pyruvate MRI-which provides real-time metabolic assessment of tissue-as an early biomarker for tissue rejection. In this preliminary study, we used μCT-derived parameters and HP 13 C MR-derived biomarkers to predict rejection in an orthotopic left lung transplant model in both allogeneic and syngeneic rats. On day 3, the normalized lung density-a parameter that accounts for both lung volume (mL) and density (HU)-was -0.335 (CI: -0.598, -0.073) and - 0.473 (CI: -0.726, -0.220) for the allograft and isograft, respectively (not significant, 0.40). The lactate-to-pyruvate ratios-derived from the HP 13 C MRI-for the allograft and isograft were 0.200 (CI: 0.161, 0.240) and 0.114 (CI: 0.074, 0.153), respectively (significant, 0.020). Both techniques showed tissue rejection on day 7. A separate sub-study revealed CD8+ cells as the primary source of the lactate-to-pyruvate signal. Our study suggests that hyperpolarized (HP) [1-13 C] pyruvate MRI is a promising early biomarker for tissue rejection that provides metabolic assessment in real time based on changes in cellularity and metabolism of lung tissue and the infiltrating inflammatory cells, and may be able to predict tissue rejection earlier than X-ray/CT.

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATPchannel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1−/−lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1−/−PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1−/−mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1−/−lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled35S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [35S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [35S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50–60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [35S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [35S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of125I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of125I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of125I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

Peroxiredoxin 6 (Prdx6), a bifunctional protein with phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid turnover. Prdx6 has been localized to both cytosol and lamellar bodies (LB) in lung epithelium, and its organellar targeting sequence has been identified. We propose that Prdx6 LB targeting facilitates its role in the metabolism of lung surfactant phosphatidylcholine (PC). Ser-32 has been identified as the active site in Prdx6 for aiPLA2 activity, and this activity was abolished by the mutation of serine 32 to alanine (S32A). However, aiPLA2 activity was unaffected by mutation of serine 32 in Prdx6 to threonine (S32T). Prdx6 protein expression and aiPLA2 activity were normal in the whole lung of a "knock-in" mouse model carrying an S32T mutation in the Prdx6 gene but were absent from isolated LB. Analyses by proximity ligation assay in lung sections demonstrated the inability of S32T Prdx6 to bind to the chaperone protein, 14-3-3ϵ, that is required for LB targeting. The content of total phospholipid, PC, and disaturated PC in lung tissue homogenate, bronchoalveolar lavage fluid, and lung LB was increased significantly in Prdx6-S32T mutant lungs, whereas degradation of internalized [(3)H]dipalmitoyl-PC was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for LB Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine.

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O(2)). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA(2) activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA(2) activity each contribute to the recovery process.

Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1−/−) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from −/− mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results ( 47 ). Lipid analysis of the lung tissue of −/− mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominately due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the −/− mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of −/− mice lungs, measured using intratracheal installation of3H-PL liposomes. Compared with WT mice, the Abca1−/−mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function.

Peroxiredoxin 6 (Prdx6), a peroxidase that also expresses phospholipase A2 (aiPLA2) activity, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosolic and acidic (lamellar body [LB] and lysosomal) compartments in lung epithelium. We propose that Prdx6 LB targeting facilitates its role in the degradation and remodeling of lung surfactant phosphatidylcholine. Although, we have previously shown that mutation of serine 32 (S32) to alanine abolishes Prdx6 aiPLA2 activity, we now show that serine to threonine mutation has no effect on either Prdx6 peroxidase or aiPLA2 activity. Since S32 is a necessary component of the amino acid motif that mediates Prdx6 LB targeting, we studied the effect of the S32T mutation on Prdx6 LB localization. In a “knock-in” mouse model carrying an S32T mutation in the Prdx6 gene, there was phospholipid (PL) accumulation in the lung and a marked decrease in aiPLA2 activity in LB. Prdx6 was absent f...

We concluded earlier that endothelial mechanotransduction with ischemia in the lung activates a signaling cascade comprised of KATP channel closure that leads to NOX2 activation and reactive oxygen...

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking.125I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

Prdx6 is a multi-functional protein that expresses peroxidase, phospholipase A2 (PLA2) and LPC acyltransferase activities. While the three activities of Prdx6 contribute to either the scavenging of lipid hydroperoxides or to the repair of peroxidized membranes, the PLA2 activity of Prdx6 (aiPLA2) is also required for NOX2 activation in pulmonary microvascular cells (PMVECs), alveolar macrophages and polymorphonuclear neutrophils. During sepsis-induced acute lung injury (ALI), NOX2 is activated generating superoxide radicals that contribute to vascular inflammation. To test whether inactivation of aiPLA2 ameliorates sepsis-induced ALI, we used a knockin mouse model (Prdx6-D140A) that lacks aiPLA2 activity due to a mutation of a key member of the PLA2 catalytic triad. Prdx6-D140A mice treated IP with LPS (10 mg/Kg) exhibited increased survival rates compared to WT mice. Total nucleated cells in BALF, VCAM-1 expression, and lung permeability increased after 24 h treatment with LPS in WT but not in Prdx6-D140A knockin mice. In vitro, intracellular hydrogen peroxide generation increased after LPS treatment (1 ug/mL for 16h) in PMVECs stably-expressing the HyPer-Cyto sensor. Increased H2O2 generation was prevented by pre-treatment with MJ33, a pharmacological inhibitor of aiPLA2 activity. These results show that aiPLA2 is needed for LPS-induced oxidant generation in PMVECs and that genetic inactivation aiPLA2 reduces LPS-induced inflammation, increased vascular permeability, and mortality in an animal model suggesting that aiPLA2 could be a target for prevention or treatment of sepsis-induced ALI.

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [3H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [3H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [3H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A −/− lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A −/− mouse. With secretagogue (8-bromoadenosine 3′,5′-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A −/− lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A −/− lung. Inclusion of SP-A with instilled liposomes served to “rescue” the SP-A −/− lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.

The mechanisms used by alveolar type I pneumocytes for maintenance of the lipid homeostasis necessary to sustain these large squamous cells are unknown. The processes may involve the ATP-binding cassette transporter A1 (ABCA1), a transport protein shown to be crucial in apolipoprotein A-I (apoA-I)-mediated mobilization of cellular cholesterol and phospholipid. Immunohistochemical data demonstrated the presence of ABCA1 in lung type I and type II cells and in cultured pneumocytes. Type II cells isolated from rat lungs and cultured for 5 days in 10% serum trans-differentiated toward cells with a type I-like phenotype which reacted with the type I cell-specific monoclonal antibody VIIIB2. Upon incubation of the type I-like pneumocytes with agents that up-regulate the ABCA1 gene (9-cis-retinoic acid [9cRA] and 22-hydroxycholesterol [22-OH, 9cRA/22-OH]), ABCA1 protein levels were enhanced to maximum levels after 8 to 16 hours and remained elevated for 24 hours. In the presence of apoA-I and 9cRA/22-OH, efflux of radioactive phospholipid and cholesterol from pneumocytes was stimulated 3- to 20-fold, respectively, over controls. Lipid efflux was inhibited by Probucol. Sucrose density gradient analysis of the media from stimulated cells incubated with apoA-I identified heterogeneous lipid particles that isolated at a density between 1.063 and 1.210 g/ml, with low or high apoA-I content. Thus, pneumocytes with markers for the type I phenotype contained functional ABCA1 protein, released lipid to apoA-I protein, and were capable of producing particles resembling nascent high-density lipoprotein, indicating an important role for ABCA1 in the maintenance of lung lipid homeostasis.

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.