Michael Cutaia

Effect of hypoxic exposure on Na+/H+antiport activity, isoform expression, and localization in endothelial cells

Little is known about the effects of prolonged hypoxic exposure on membrane ion transport activity. The Na+/H+antiport is an ion transport site that regulates intracellular pH in mammalian cells. We determined the effect of prolonged hypoxic exposure on human pulmonary arterial endothelial cell antiport activity, gene expression, and localization. Monolayers were incubated under hypoxic or normoxic conditions for 72 h. Antiport activity was determined as the rate of recovery from intracellular acidosis. Antiport isoform identification and gene expression were determined with RT-PCR and Northern and Western blots. Antiport localization and F-actin cytoskeleton organization were defined with immunofluorescent staining. Prolonged hypoxic exposure decreased antiport activity, with no change in cell viability compared with normoxic control cells. One antiport isoform [Na+/H+exchanger isoform (NHE) 1] that was localized to the basolateral cell surface was present in human pulmonary arterial endothelial cells. Hypoxic exposure had no effect on NHE1 mRNA transcript expression, but NHE1 protein expression was upregulated. Immunofluorescent staining demonstrated a significant alteration of the F-actin cytoskeleton after hypoxic exposure but no change in NHE1 localization. These results demonstrate that the decrease in NHE1 activity after prolonged hypoxic exposure is not related to altered gene expression. The change in NHE1 activity may have important consequences for vascular function.

Sphingomyelin synthase 2 (SMS2) deficiency attenuates LPS-induced lung injury

Sphingomyelin synthase (SMS) catalyzes the synthesis of sphingomyelin (SM) and is required for maintenance of plasma membrane microdomain fluidity. Of the two isoforms of mammalian SMS, SMS1 is mostly present in the trans-Golgi apparatus, whereas SMS2 is predominantly found at the plasma membrane. SMS2 has a role in receptor mediated response to inflammation in macrophages, however, the role of SMS2 in vascular permeability, pulmonary edema, and lung injury have not been investigated. To define the role of SMS activation in lung injury, we utilized a lipopolysaccharide (LPS)-induced lung edema model. SMS activity was measured and correlated with the severity of lung injury. Within 4 h of LPS treatment, SMS activity was increased significantly and remained upregulated up to 24 h. Comparison of LPS-induced lung injury in SMS2 knockout (SMS2(-/-)) and wild-type littermate control mice showed that inflammation, cytokine induction, and lung injury were significantly inhibited in SMS2(-/-) mice. Our results suggest that a deficiency of SMS2 can diminish the extent of pulmonary edema and lung injury. Furthermore, we show that depletion of SMS2 was sufficient to decrease MAP kinase-JNK activation, severity of LPS-induced pulmonary neutrophil influx, and inflammation, suggesting a novel role of SMS2 activation in lung injury.

A novel approach to measuring activity in chronic obstructive pulmonary disease: using 2 activity monitors to classify daily activity.

PURPOSE Patients with chronic obstructive pulmonary disease with a low profile of daily activity have poor health outcomes. Although walking is a common activity, it may not be the most relevant physical activity to measure in this population. It was the purpose of this study to determine the accelerometer-defined thresholds that discriminate a range of daily activities and use these thresholds to assess activity profiles among stages of disease severity. METHODS Subjects with chronic obstructive pulmonary disease (N = 57) completed a standardized sequence of activities that comprised sitting, standing, and walking while wearing an accelerometer on the waist and an actigraph on the wrist. Using a calibration procedure, accelerometer output was translated into speeds. Speeds were estimated for each interval in the testing sequence. Walking and nonacceleration thresholds were derived from the intervals to define 4 activity categories: walking, slow/intermittent walking, active-not-walking, and rest. Subjects wore the 2 devices for 2 days. Accelerometer output was then classified into 1 of the activity categories. Percent time spent in activity categories and speeds generated were compared among Global Initiative for Obstructive Lung Disease (GOLD) stages. RESULTS The waist-worn accelerometer accurately estimated speeds. Speed thresholds for walking and nonacceleration were 0.70 mph and 0.25 mph, respectively. Among GOLD stages, those with more severe obstruction spent less time in the walking categories and generated slower speeds. CONCLUSIONS The accuracy of these methods to detect a range of physical activities enhances the utility of accelerometers in comparing daily activity in sedentary populations. Measurements of the more subtle activities offer an appealing new area of study.

Studies on the mechanism of short-term regulation of pulmonary artery endothelial cell Na/K pump activity

The Na/K pump is critically important in maintenance of cell homeostasis in the face of injury. Little is known about the regulation of endothelial cell Na/K-pump activity. We previously reported that short-term (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery endothelial cells (BPAECs). In this study we investigated the mechanism of oxidant-induced increases in endothelial Na/K-pump activity, focusing on short-term modulation of alpha1-pump subunit. By using immunofluorescence microscopy and confocal scanning laser microscopy, we found alpha1 subunit on both apical and basal aspects of BPAECs without polarized distribution. Short-term (30-minute) incubation of PAEC monolayers with H2O2 (1 mmol/L) did not change the relative amounts of alpha1 subunit in membrane fractions, as assessed by immunoblotting. Phosphorylation of the alpha1 subunit also was not affected by H2O2 treatment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H2O2 has been reported to increase PKC activity of endothelial cells, we determined the effects of inhibition and activation of protein kinase C (PKC) on Na/K-pump activity quantitated as ouabain-inhibitable uptake of 86Rb. We also determined the effects of PKC activation and inhibition on H2O2-induced increases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump activity over a 30-minute period in intact monolayers. Inhibition or depletion of PKC did not prevent H2O2-induced increases in pump activity. These results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H2O2 are not mediated by activation of PKC or by changes in the expression or phosphorylation of alpha1 subunit.

Role of Sphingomyelin Synthesis in Pulmonary Endothelial Cell Cytoskeletal Activation and Endotoxin-Induced Lung Injury

Sphingomyelin (SM), a major sphingolipid in the lipid raft microdomains of the cell membrane, is synthesized by plasma membrane-bound sphingomyelin synthase 2 (SMS2). SMS2 is required for the maintenance of plasma membrane microdomain fluidity and receptor-mediated responses to inflammation in macrophages. However, the exact mechanism of SMS2 activation in endothelial barrier disruption and lung injury is not fully understood. To define the role of SMS activation in lung injury, we hypothesized that the inhibition of SM synthesis may provide protection against acute lung injury (ALI) by preserving endothelial barrier function. Using SMS2-silencing RNA (siRNA) treatment in human pulmonary endothelial cells (HPAECs) and tricyclodecan-9-yl-xanthogenate (D609), a competitive inhibitor of SMS, and phosphatidylcholine-specific phospholipase C in a murine model of bacterial LPS injury, we studied the role of sphingomyelin synthesis in ALI. Results show that pretreating mice with D609 significantly attenuated LPS-induced lung injury, as measured by a significant decrease in wet to dry ratio, bronchoalveolar lavage fluid cell and protein counts, and myeloperoxidase activity in lung tissue. Similarly, LPS-induced endothelial barrier disruption was significantly reduced in HPAECs pretreated with D609 or SMS2 siRNA, as demonstrated by an increase in paracellular integrity on an FITC-dextran assay, by the inhibition of LPS-induced stress fibers, and by the formation of cortical actin rings and lamellipodia at the periphery. These results indicate that D609 attenuates LPS-mediated endothelial barrier dysfunction and lung injury in mice through inhibition of SMS, suggesting a novel and essential role of SMS inhibition in modulating endothelial barrier integrity via actin cytoskeletal activation, with a potential therapeutic role in ALI.

Differences in nucleotide effects on intracellular pH, Na+/H+ antiport activity, and ATP-binding proteins in endothelial cells

SummaryBovine (BPAEC) and human (HPAEC) pulmonary artery endothelial cell monolayers were incubated with either ATP, ATP analogues, or UTP, followed by measurement of intracellular pH (pHi) and the rate of recovery from acidosis. ATP increased baseline pHi and the rate of acid recovery in BPAEC. This response was inhibited by the amiloride analogue, methyisobutylamiloride, demonstrating that activation of the Na+/H+ antiport was responsible for the increase in baseline pHi and the recovery from acidosis. This response had the features of both a P2Y and P2U purinergic receptor, based on the responses to a series of ATP analogues and UTP. In contrast, none of the nucleotides had any significant effect on pHi and Na+/H+ antiport activity in HPAEC. This difference in the response to extracellular nucleotides was not due to a difference in ATP metabolism between cell types, since the ectonucleotidase-resistant analogue, ATPγS, also had no effect on HPAEC. Analogues of cAMP had no effect on pHi or acid recovery in either cell type. Incubation of BPAEC and HPAEC with the photoaffinity ligand [32P] 8-AzATP indicated that both BPAEC and HPAEC possess an ATP-binding protein of 48 kDa. However, BPAEC exhibited an additional binding protein of 87 kDa. Thus, the contrasting response to extracellular ATP between bovine and human pulmonary artery endothelial cells may be related to differences in the signal transduction pathway leading to antiport activation, including different ATP-binding sites on the cell membrane.

Detecting Motor Vehicle Travel in Accelerometer Data

Abstract Chronic Obstructive Pulmonary Disease (COPD) frequently has a significant impact on patients’ everyday activity. Because of this, accurate measurement of daily activity is of particular interest. Although accelerometers are an objective means of measuring daily activity, these devices sense vibrations and erroneously score motor vehicle travel (MVT) as moderate physical activity. It is the objective of this study to develop a new method to analyze accelerometry data that would accurately classify MVT as non-acceleration, or sitting/standing. As sitting/standing has a different pattern of count-to-count variability than walking, we hypothesized that a rolling standard deviation (RSD), which is a measurement of volatility in the data, would more accurately classify periods of MVT than analysis based on activity counts alone. Twenty-two subjects with COPD were studied. A training set of 15% of the dataset was used to establish an RSD-threshold during MVT based on the upper 95%-confidence interval. The accuracy of the RSD thresholds were tested and presented as sensitivity, specificity and receiver operating curves. Results demonstrated high sensitivity and specificity suggesting that the RSD not only accurately classified MVT, but had a low rate of misclassification. The RSD analysis scored more MVT as sitting/standing than assessment by VMU alone. The accuracy of accelerometers to define the profile of daily activity in sedentary populations, such as those with COPD, is greatly improved.

Tracheal squamous cell carcinoma treated endoscopically.

Malignant primary tracheal tumors are extremely rare. The most common malignant primary tracheal tumors are squamous cell carcinoma and adenoid cystic carcinomas. In this brief report, we describe a patient who presented with a primary papillary squamous cell carcinoma in-situ at multiple areas in the trachea with a significant airway obstruction. Our case was successfully managed using a combination of electrocautery and argon photocoagulation for endotracheal ablation of the tumor and adjuvant external beam radiotherapy.

Defining expanded areas in EBUS sampling: EBUS guided trans- and intra-pulmonary artery needle aspiration, with review of transvascular EBUS

Endobronchial Ultrasound‐guided Transbronchial Needle Aspiration (EBUS‐TBNA) has revolutionized the diagnostic approach to mediastinal diseases. Lesions located lateral to the pulmonary artery (trans‐PA, Station 5), or in the lumen of the PA (intra‐PA) are in the ‘blind‐spot’ of EBUS.

Incidental carcinoid tumor next to vascular malformation.

Dedicated linear endobronchial ultrasound (EBUS) is an established modality in sampling central pulmonary lesions, mediastinal and hilar lymph nodes to diagnose and stage lung malignancies as well as evaluate a number of inflammatory conditions. In addition, power Doppler modality of EBUS allows for vascular evaluation. We describe a case where EBUS visualized a submucosal lesion within a vascular malformation, which was eventually proven to be a carcinoid tumor. Computed tomographic scan of the chest failed to differentiate the 2 structures and bronchoscopy showed no endobronchial component. This case underscores the added diagnostic capabilities of endobronchial Doppler ultrasonography. We suggest considering EBUS in investigating central opacities associated with vascular features within the lung parenchyma when technically feasible.