Rapid apoptosis in the pulmonary vasculature distinguishes non-metastatic from metastatic melanoma cells.

The presence of metastases indicates an ominous prognosis in patients with malignancies, yet the factors that distinguish metastatic from non-metastatic tumors remain poorly understood. Here we pursued the hypothesis that apoptosis in vivo would distinguish metastatic cells from non-metastatic cells and developed a novel method for observation of apoptosis induction in living cells. One hour after the infusion of metastatic or non-metastatic human melanoma or transformed rat embryo fibroblasts, arrest of tumor cells in the pulmonary vasculature was equivalent. In order to demonstrate the induction of apoptosis in living cells, we observed the translocation of cytoplasmic BAD-GFP fusion proteins to the mitochondria during apoptosis. Microscopic observation of the tumor cells transfected with BAD-GFP in isolated lung preparations after intravenous injection into nu/nu mice revealed translocation of BAD-GFP in many more of the arrested, non-metastatic melanoma or transformed rat embryo cells over 4-24 h than of the metastatic cells. TUNEL staining confirmed enhanced apoptosis by non-metastatic tumor cells after injection in vivo. Metastatic melanoma cells or metastatic embryo fibroblasts were better able to negotiate the barrier of survival in the circulation after pulmonary arrest than non-metastatic cells confirming the hypothesis that susceptibility to apoptosis after arrest in the pulmonary vasculature distinguishes metastatic from non-metastatic cells and introducing a new assay for in vivo induction of apoptosis.

Lung ischemia: endothelial cell signaling by reactive oxygen species. A progress report.

These studies using both intact lung and reconstituted cell systems have shown that pulmonary endothelial cells respond rapidly (within several seconds) to the acute cessation of perfusate flow, i.e., ischemia. These effects represent a response to the loss of shear stress and are unrelated to changes in cellular oxygenation. The immediate response is partial depolarization of the endothelial cell membrane followed by activation of endothelial NADPH oxidase and the extracellular generation of superoxide anion. Dismutation of superoxide to H2O2 generates a cell signaling molecule that results in the activation of protein kinases and transcription factors which in turn lead to NO generation and activation of endothelial cell division. The presumed physiological role of this signal cascade is the generation of a vasodilator (NO) and the formation of new capillaries in the effort to restore blood flow.

TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs.

We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K(f)) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH(2)O PIP at both 35 degrees C and 40 degrees C. Ventilation with 35 cmH(2)O PIP increased K(f) by 2.2-fold at 35 degrees C and 3.3-fold at 40 degrees C compared with baseline, but K(f) increased significantly with time at 40 degrees C with 9 cmH(2)O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in K(f). In TRPV4(-/-) knockout mice, the high PIP ventilation protocol did not increase K(f) at either temperature. We have also found that lung distention caused Ca(2+) entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4(-/-) and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4(-/-) lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.

Shear stress and endothelial cell activation.

We have shown previously that ischemia in an isolated rat lung that is normally oxygenated by continued ventilation results in lipid and protein oxidation, indicating the generation of reactive oxygen species. By using a variety of biochemical and imaging techniques, we determined that the initial response, which occurs within the first second of ischemia, is partial depolarization of the endothelial cell plasma membrane. This event is followed within several seconds by activation of endothelial NADPH oxidase and generation of superoxide anion at the extracellular surface of the cell membrane where it is dismutated to freely diffusible H2O2. Approximately 15 secs after the onset of ischemia, we detected an elevation of intracellular Ca2+ caused by its release from intracellular stores, followed by Ca2+ influx, possibly through T-type voltage-dependent Ca2+ channels. Increased nitric oxide generation through activation of endothelial nitric oxide synthase is detected after about 45 secs of ischemia. These changes (endothelial membrane depolarization, reactive oxygen species production, elevation of intracellular Ca2+ levels, and nitric oxide generation) were confirmed in isolated endothelial cells that had been adapted to shear stress in vitro. The in vitro model also demonstrates reactive oxygen species-dependent activation of nuclear factor-kappaB and activator protein-1 and that 24 hrs of ischemia results in increased cell division. These results indicate a novel cell-signaling pathway in response to loss of shear stress. The basis for these changes in endothelial function is related to mechanotransduction, i.e., altered shear stress rather than a metabolic response to ischemia. The biological function for the response may be an attempt to restore blood flow through vasodilatation and new capillary formation.

Ca2+ flux through voltage-gated channels with flow cessation in pulmonary microvascular endothelial cells.

OBJECTIVE: To investigate the role of voltage-gated Ca2+ channels in Ca2+ influx with flow cessation in flow-adapted rat pulmonary microvascular endothelial cells. METHODS: Cells were evaluated for mRNA and protein levels for major components of the voltage-gated Ca2+ channels. Ca2+ influx with flow cessation and cell membrane potential were measured in real time with fluorescent dyes. Mibefradil and nifedipine were used as inhibitors of Ca2+ channel activity. RESULTS: Voltage-gated Ca2+ channel protein and mRNA for the T-type channel were expressed at a relatively low level in endothelial cells cultured under static conditions and expression was induced significantly during flow adaptation. Flow-adapted but not control cells showed Ca2+ influx during flow cessation that was blocked by mibefradil but not by nifedipine. Ca2+ influx also was blocked by cromakalim, a KATP channel agonist. Cell membrane depolarization with flow cessation was unaffected by mibefradil. CONCLUSIONS: Rat pulmonary microvascular endothelial cells express T-type voltage-gated Ca2+ channels that are induced during adaptation to flow and are responsible for Ca2+ influx that occurs as a result of flow cessation-mediated membrane depolarization.