Abrupt cessation of lung perfusion induces a rapid endothelial response that is not associated with anoxia but reflects loss of normal shear stress. This response includes membrane depolarization, H(2)O(2) generation, and increased intracellular Ca(2+). We evaluated these parameters immediately upon nonhypoxic ischemia using fluorescence videomicroscopy to image in situ endothelial cells in isolated, ventilated rat lungs. Lungs labeled with 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS; a membrane potential probe), Amplex Red (an extracellular H(2)O(2) probe), or fluo 3-AM (a Ca(2+) indicator) were subjected to control perfusion followed by global ischemia. Endothelial di-8-ANEPPS fluorescence increased significantly within the first second of ischemia and stabilized at 15 s, indicating membrane depolarization by approximately 17 mV; depolarization was blocked by preperfusion with the K(+) channel agonist lemakalim. Increased H(2)O(2), inhibitable by catalase, was detected in the vascular space at 1-2 s after the onset of ischemia. Increased intracellular Ca(2+) was detected 10-15 s after the onset of ischemia; the initial increase was inhibited by preperfusion with thapsigargin. Thus the temporal sequence of the initial response of endothelial cells in situ to loss of shear stress (i.e., ischemia) is as follows: membrane depolarization, H(2)O(2) release, and increased intracellular Ca(2+).

In this study, we examined the hypothesis that early pulmonary metastases form within the vasculature. We introduced primary tumors in immunocompromised mice by subcutaneous injection of murine breast carcinoma cells (4T1) expressing green fluorescent protein. Isolated ventilated and perfused lungs from these mice were examined at various times after tumor formation by fluorescent microscopy. The vasculature was visualized by counterstaining with 1,1-dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine (DiI)-acetylated low-density lipoprotein. These experiments showed that metastatic cells derived by spontaneous metastases were intravascular, and that early colony formation was intravascular. The location of the tumor cells was confirmed by deconvolution analysis. This work extends our previous study 1 We recently reported direct observations of the sequence of events in pulmonary metastasis after injection of fluorescent tumor cells into the venous circulation using fluorescent microscopy of isolated perfused lungs. 1 Because endothelium has receptors for oxidized or acetylated low-density lipoprotein (LDL), the pulmonary endothelium can be precisely visualized in these preparations through infusion of DiI-acetylated LDL. Because the lung is translucent, these methods allow high-resolution imaging of the microvasculature of the lungs at up to 100-m depth beneath the pleural surface and enable the precise localization of the tumor cells. We used these methods to show in a mouse experimental metastasis model that tumor cells attach to the pulmonary endothelium and proliferate intravascularly.We have also demonstrated that differences in apoptosis of tumor cells in vivo correlated to differences in metastatic potential. 1-3 Based on these observations, we proposed a new model for pulmonary metastasis in which endothelium-attached tumor cells that survived the initial apoptotic stimuli proliferate intravascularly. A principal tenet of this new model is that extravasation of tumor cells is not a prerequisite for metastatic foci formation. [1][2][3] Because the initial experiments were based on intravenous injection of tumor cells, it was possible that the results were an artifact of this mode of introduction of the tumor cells or of the relatively large numbers of cells infused simultaneously. To determine whether our observations in an experimental model of metastasis were valid for cells that metastasize spontaneously from a primary tumor, we have tested our hypothesis using a spontaneous metastasis model. Our previous results were obtained using fibrosarcoma cells, so we sought to extend this hypothesis to carcinoma cells by using the breast carcinoma cell line 4T1. 4 The results indeed indicate that extravasation is also rare after spontaneous metastasis. These observations confirmed that metastasizing tumor cells proliferate intravascularly within the lung. This suggests that drug discovery efforts could be directed to blocking the metastatic cascade at the steps of the initial Supported by the Susan G. Komen Bre...

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N G -nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ϳ55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. In the lung, metastatic neoplasms are the most common malignant tumors. Their formation usually involves hematogenous seeding of metastatic cancer cells into the lung from remote primary tumors. Interactions between intravascular cancer cells and the endothelium are important determinants of metastatic outcome. 1,2 For example, the expression of constitutive and inducible microvascular adhesion molecules, and the release of reactive oxygen species (NO, O 2 Ϫ , and H 2 O 2 ) by endothelial cells or cancer cells can regulate the mechanisms that govern the metastatic process, including cancer cell adhesion and arrest, 3 the production of endothelial matrix metalloproteinases, 4 and cancer cell apoptosis. 5 Evidence from in vitro and in vivo studies has shown that reactive oxygen and nitrogen species can be cytotoxic to neoplastic cells 6 -9 and reduced their adhesion to postcapillary venules. 10 In vivo, we have recently demonstrated that the arrest of intravascular B16F1 melanoma cells in the liver induces the rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. 5 Because pulmonary endothelial cells generate NO in response to shear stress, 11 we have postulated that there is a comparable cytotoxic mechanism in the lung.Here we provide data showing that the arrest of B16F1 melanoma cells in the pulmonary circulation of wild-type C57B1/6 mice (WT mice) induces the ...

Hereditary spastic paraplegia (HSP) is a neurodegenerative disease characterized by lower-limb spasticity, hyperreflexia, progressive spastic gait abnormalities, and an extensor-plantar response. It is genetically very heterogeneous, with 28 Human Genome Organisation (HUGO)-approved IDs in the database (last search: August 8, 2004). Following the identification of the SPG6 gene, NIPA1, we have identified two novel mutations, c.316G>C and c.316G>A, in two independent Chinese families linked to the SPG6 locus. These two mutations would cause a p.G106R substitution, and cosegregated with the disease. Structural predictions suggest that p.G106 is located in the third transmembrane domain of the protein, and that the mutant p.G106R disrupts this structure, causing the intramembrane loop to descend into the cytoplasm. Our results identify two novel mutations responsible for HSP and suggest that c.316 of theNIPA1 gene may be a mutational hotspot.

Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca 2؉ . In situ endothelial generation of NO and changes in intracellular Ca 2؉ in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca 2؉ probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca 2؉ due to both influx of extracellular Ca 2؉ and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca 2؉ -, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.Nitric oxide (NO) 1 is a potent regulator of vascular tone in systemic and pulmonary vessels and plays an important role in cellular signaling and respiration (1-4). This mediator is generated by endothelial cells in response to agonist stimulation and also as a response to increased shear stress (5). Although the effect of increase in flow on endothelial cell NO generation has been characterized, the effect of abrupt cessation of shear stress (i.e. acute ischemia as in pulmonary embolism or donor lung isolation for transplantation) on pulmonary endothelial NO generation in situ is unknown.Endothelial cells in situ are constantly exposed to shear stress associated with blood flow and thus become flowadapted. These cells respond with an increase in cytosolic Ca 2ϩ and generation of reactive oxygen species when flow is stopped but oxygenation is maintained (6, 7). A similar response with reactive oxygen species generation subsequent to the abrupt cessation of flow has been demonstrated with endothelial cells adapted to flow in vitro (8). We hypothesized that the basis for this early response is mechanotransduction related to removal of endothelial cell shear stress (9). Increased intracellular Ca 2ϩ is known to activate endothelial nitric-oxide synthase (eNOS) resulting in increased NO generation and has been demonstrated in cells exposed to increased shear stress (10,11). In these nonadapted endothelial cells, shear-stress-induced NOS activation was biphasic, with an initial Ca 2ϩ -dependent phase and a second, sustained phase that was Ca 2ϩ -independent and phosphorylation-dependent (10 -12). In this study, we evaluated whether the increased intracellular Ca 2ϩ associated with the early response to flow cessation leads to increased NO generation. This paradigm constitu...