PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium.

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 KATP channel, 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.

Rab38 targets to lamellar bodies and normalizes their sizes in lung alveolar type II epithelial cells.

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.

Mechanotransduction drives post ischemic revascularization through K(ATP) channel closure and production of reactive oxygen species.

AIMS: We reported earlier that ischemia results in the generation of reactive oxygen species (ROS) via the closure of a K(ATP) channel which causes membrane depolarization and NADPH oxidase 2 (NOX2) activation. This study was undertaken to understand the role of ischemia-mediated ROS in signaling. RESULTS: Angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) was studied in vitro and in the hind limb in vivo. Flow adapted PMVEC injected into a Matrigel matrix showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of K(ATP) channels or the NOX2. Blocking of hypoxia inducible factor-1 alpha (HIF-1α) accumulation completely abrogated the tube formation in wild-type (WT) PMVEC. With ischemia in vivo (femoral artery ligation), revascularization was high in WT mice and was significantly decreased in mice with knockout of K(ATP) channel and in mice orally fed with a K(ATP) channel agonist. In transgenic mice with endothelial-specific NOX2 expression, the revascularization observed was intermediate between that of WT and knockout of K(ATP) channel or NOX2. Increased HIF-1α activation and vascular endothelial growth factor (VEGF) expression was observed in ischemic tissue of WT mice but not in K(ATP) channel and NOX2 null mice. Revascularization could be partially rescued in K(ATP) channel null mice by delivering VEGF into the hind limb. INNOVATION: This is the first report of a mechanosensitive ion channel (K(ATP) channel) initiating endothelial signaling that drives revascularization. CONCLUSION: The K(ATP) channel responds to the stop of flow and activates signals for revascularization to restore the impeded blood flow.

Functional interaction of glutathione S-transferase pi and peroxiredoxin 6 in intact cells.

Peroxiredoxin 6 (Prdx6) is a 1-Cys member of the peroxiredoxin superfamily that plays an important role in antioxidant defense. Glutathionylation of recombinant Prdx6 mediated by π glutathione S-transferase (GST) is required for reduction of the oxidized Cys and completion of the peroxidatic catalytic cycle in vitro. This study investigated the requirement for πGST in intact cells. Transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increased phospholipid peroxidase activity as measured in cell lysates and protected intact cells against a peroxidative stress. siRNA knockdown indicated that this increased peroxidase activity was Prdx6 dependent. Interaction between πGST and Prdx6, evaluated by the Duolink Proximity Ligation Assay, was minimal under basal conditions but increased dramatically following treatment of cells with the oxidant, tert-butyl hydroperoxide. Interaction was abolished by mutation of C47, the active site for Prdx6 peroxidase activity. Depletion of cellular GSH by treatment of cells with buthionine sulfoximine had no effect on the interaction of Prdx6 and πGST. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST and that the interaction plays an important role in regenerating the peroxidase activity of Prdx6.

Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro.

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.