Abu-Bakr Al-Mehdi

Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription.

Reactive oxygen species generated by mitochondria that redistribute near the nucleus promote transcriptional responses to hypoxia. Mitochondria for Transcription A key response to reduced oxygen tension, a condition referred to as hypoxia, involves the hypoxia-inducible factor (HIF) family of transcription factors. During hypoxia, HIF-1α translocates to the nucleus to activate genes involved in adapting to oxygen deprivation. Al-Mehdi et al. showed that the transcriptional response to hypoxia was accompanied by the subcellular redistribution of mitochondria around the nucleus. Reactive oxygen species produced by the redistributed mitochondria caused oxidative modification of the promoter regions of HIF-1 target genes, such as that encoding vascular endothelial growth factor (VEGF). The introduction of oxidative modifications in these promoters enhanced HIF-1α association and gene expression. Because the presence of hypoxia in solid tumors is an indicator of poor prognosis, understanding the details of the transcriptional response to hypoxia may provide new targets for the therapeutic treatment of solid tumors. Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA–mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.

Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.

Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells

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 approximately 55% 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.

Ca2+ entry via α1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

An Oxidative DNA "Damage" and Repair Mechanism Localized in the VEGF Promoter is Important for Hypoxia-induced VEGF mRNA Expression

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.

Nuclear protein-induced bending and flexing of the hypoxic response element of the rat vascular endothelial growth factor promoter

Bending and flexing of DNA may contribute to transcriptional regulation. Because hypoxia and other physiological signals induce formation of an abasic site at a key base within the hypoxic response element (HRE) of the vascular endothelial growth factor (VEGF) gene (FASEBJ. 19, 387–394, 2005) and because abasic sites can introduce flexibility in model DNA sequences, in the present study we used a fluorescence resonance energy transfer‐based reporter system to assess topological changes in a wild‐type (WT) sequence of the HRE of the rat VEGF gene and in a sequence harboring a single abasic site mimicking the effect of hypoxia. Binding of the hypoxia‐inducible transcriptional complex present in hypoxic pulmonary artery endothelial cell nuclear extract to the WT sequence failed to alter sequence topology whereas nuclear protein binding to the modified HRE engendered considerable sequence flexibility. Topological effects of nuclear proteins on the modified VEGF HRE were dependent on the transcription factor hypoxia‐induc‐ible factor‐1 and on formation of a single‐strand break at the abasic site mediated by the coactivator, Ref‐1/ Ape1. These observations suggest that oxidative base modifications in the VEGF HRE evoked by physiological signals could be a precursor to single‐strand break formation that has an impact on gene expression by modulating sequence flexibility.— Breit, J. F., Ault‐Ziel, K., Al‐Mehdi, A.‐B., Gillespie, M. N. Nuclear protein‐induced bending and flexing of the hypoxic response element of the rat vascular endothelial growth factor promoter. FASEB J. 22, 19–29 (2008)

Early incorporated endothelial cells as origin of metastatic tumor vasculogenesis

Vascularization of solid tumors is thought to occur by sprouting or intussusceptive angiogenesis, co-option of existing vessels, and vasculogenic mimicry after the onset of tumor hypoxia, when the tumor radius exceeds the oxygen diffusion distance. In contrast, here we show that individual endothelial cells that are incorporated into pre-hypoxic tumors give rise to tumor blood vessels via vasculogenesis. Small metastatic lung tumor sections obtained after tail-vein injection of a syngeneic breast cancer cell line in the nude mice were labeled with antibodies against endothelial cell markers. Immunofluorescence showed the incorporation and mixed growth of CD31-, Tie-2-, and CD105-positive endothelial cells in tumors with radii less than oxygen diffusion distance and subsequent development of blood vessels from these early-incorporated endothelial cells. This observation lays the foundation of a novel vasculogenic paradigm of tumor vascularization, where incorporation of endothelial cells and their growth among tumor cells occur before the onset of core hypoxia in lung metastatic tumors.

Increased depth of cellular imaging in the intact lung using far-red and near-infrared fluorescent probes.

Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834) to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm) was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8 ± 0.7  μm) than for GFP (20 ± 0.5  μm; p = 0.008; n = 50), corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4 ± 1  μm from the pleural surface) compared with GFP (30.1 ± 0.5  μm; p = 0.01; n = 50). Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.

Mitochondria in hypoxic pulmonary vasoconstriction: potential importance of compartmentalized reactive oxygen species signaling.

About 15 years ago, Paul Schumacker and his then student, Nav Chandel, reported that in cultured Hep3b cells hypoxia caused mitochondrial production of reactive oxygen species (ROS) that were necessary for accumulation of the master transcriptional regulator in hypoxia, Hif1, and attendant gene expression (1). Since then—and including an important contribution in this issue of the Journal (pp. 424–432) (2)—Greg Waypa and the Schumacker research team have shown that specific disruption of mitochondrial complex III function by genetic deletion of the nuclear gene encoding the Riske iron-sulfur protein (RISP) inhibits hypoxia-induced ROS generation, calcium mobilization, and acute pulmonary vasoconstriction. The current work is significant for at least two reasons. First, it translates previous observations in cultured pulmonary arterial smooth muscle cells (PASMCs) to intact RISP-deficient mice, thus confirming in an integrated system that complex III is indeed a source of hypoxia-induced ROS generation. And second, the current article continues an evolving theme that ROS-mediated signaling is compartmentalized. Regarding the first issue, the murine reagent described by Waypa and colleagues should enable more detailed studies of the links between complex III–derived ROS and pathophysiologic processes that can be examined only in an intact model. Among many possible uses of the model, several seem especially pertinent. For example, it may now be possible to determine whether the long-appreciated suppression of localized hypoxic vasoconstriction and dysregulation of ventilation–perfusion matching in sepsis (3) are associated with defective signaling at the level of mitochondrial complex III. In addition, and as the authors point out, although the available data support the view that complex III–derived ROS are important for the calcium mobilization underlying acute hypoxic vasoconstriction, the question of whether a similar ROS-dependent pathway drives sustained pulmonary vasoconstriction and vascular remodeling in chronic hypoxic pulmonary hypertension should now be amenable to resolution. Finally, the article by Waypa and colleagues raises intriguing questions about the cellular basis of hypoxic pulmonary vasoconstriction. Whereas the current findings support the concept that the PASMC is both a sensor and an effector of hypoxic pulmonary vasoconstriction (3), Wang and coworkers, using multiple strategies to inhibit connexin 40 (Cx40)-mediated gap junctional signaling in intact mice, recently presented evidence that the vasoconstrictor response to alveolar hypoxia is initiated by depolarization of pulmonary capillary endothelial cells (4). In their paradigm, identified as “out of the box” in an accompanying editorial (5), the pulmonary microvascular endothelium, not the pulmonary arterial smooth muscle, functions as the cellular oxygen sensor that initiates a signal conducted retrogradely via endothelial Cx40-containing gap junctions to activate smooth muscle contraction in upstream muscular arteries. Some of the observations reported by Waypa and colleagues may bear on this divergence of evidence. They noted in cultured PASMCs that hypoxia caused only a transient increase in cytosolic calcium that was inhibited by RISP depletion, whereas in small arteries observed in precision-cut lung slices, the RISP-sensitive calcium response was sustained over the course of hypoxic exposure. Perhaps these temporal differences in cytosolic calcium accumulation reflect the contribution of endothelial cells to regulation of hypoxic vasoconstriction in the intact lung tissue that does not occur in cultured PASMCs. Taking this notion one step further, it is reasonable to consider whether the molecular mechanism of the hypoxia-induced depolarization of the pulmonary capillary endothelial cell is similar to that identified in the PASMC; that is, is the endothelial cell depolarization triggered by increased mitochondrial ROS production? Hopefully, the sophisticated strategies and reagents used by the Schumacker (2) and Kuebler (4) labs can be combined to address these questions. The article by Waypa and colleagues also contributes to the evolving concept that ROS-dependent signaling is highly compartmentalized. Using reduction-oxidation–sensitive green fluorescent protein (roGFP) redox probes targeted to the mitochondrial matrix, intermembrane space, and cytoplasm, they found that hypoxia exerted divergent effects on the redox status of these cellular compartments; whereas the mitochondrial matrix was progressively reduced, the intermembrane space and cytosol became more oxidized. This compartmentalized pattern of oxidant stress makes sense; the hypoxia-induced oxidant stress originating at complex III is “vectored” away from the mitochondrial matrix where oxidative damage to the sensitive mitochondrial genome could be expected to disrupt mitochondrial transcription, possibly triggering a bioenergetic crisis and/or cell death (6). And as shown by Waypa and coworkers and discussed below, the oxidant stress in the cytosol triggers calcium accumulation necessary for hypoxic pulmonary vasoconstriction. Because the roGFP used in the study by Waypa and colleagues was diffusely distributed throughout the cytoplasm, it was not possible to determine if the hypoxia-induced, mitochondria-dependent oxidant stress was more or less prominent in specific cytoplasmic domains. This becomes an interesting issue because it is widely appreciated that mitochondria are motile organelles whose distribution has the potential to determine their functional activities (7). For example, kinesin-dependent movement of mitochondria to a submembrane region in close proximity to the “immunologic synapse” in activated immune cells increases local calcium buffering capacity to sustain transmembrane calcium influx through calcium release–activated membrane calcium channels (8). More germane to the focus of the study by Waypa and coworkers, Al-Mehdi and colleagues recently showed in hypoxic pulmonary artery endothelial cells that dynein-driven perinuclear clustering of ROS-producing mitochondria creates a nuclear oxidant stress (9). This nuclear oxidant stress leads to oxidative base modifications in promoter sequences of hypoxia-inducible genes that seem to be important for transcriptional activation. Noted but not pursued in this latter report was the observation that perinuclear mitochondrial clustering was accompanied by a diminution in the peripheral cytosolic density of mitochondria. Putting these findings in the context of Waypa and colleagues’ article, it is tempting to speculate that acute hypoxia-induced mitochondrial ROS production interacts with time-dependent changes in mitochondrial distribution to create signaling microdomains (Figure 1). One critical domain could be at the mitochondrial–sarcoplasmic reticulum (SR) interface, where local ROS might function to regulate SR calcium release and cytosolic calcium accumulation. By contrast, the relative diminution of mitochondria in the vicinity of the cell membrane could serve to create a local environment relatively deficient in mitochondria-derived signaling molecules or functions, thereby impacting the operation of plasma membrane ion channels and other processes. Figure 1. Signal compartmentalization by source translocation. Mitochondria (purple), being motile organelles, can create localized signaling environments by clustering to specific cellular compartments. In normoxia, mitochondria in pulmonary arterial smooth muscle ... The mechanism underlying the reported link between mitochondrial-derived oxidant stress and the hypoxia-induced cytosolic calcium accumulation required for pulmonary vasoconstriction is not understood. It is possible, however, that a compartmentalization of the oxidant stress may contribute to these regulatory processes. In this regard, experiments in cultured PASMCs suggest that hypoxia may induce SR calcium release by causing ROS-mediated dissociation of FK506 binding protein 12.6 from ryanodine receptor 2 (10, 11). Another contributory mechanism might be that perimitochondrial ROS impair mitochondrial calcium sequestration, as reported for carotid glomus cells (12), thereby exaggerating cytosolic calcium accumulation triggered by SR calcium release. In this scenario, SR calcium release and mitochondrial calcium uptake would be reciprocally regulated by complex III–generated ROS. The concept of compartmentalized regulation of ROS signaling in hypoxia as advanced above and inferred from the data of Waypa and colleagues is far from proven. However, we think that it makes intuitive sense. After all, ROS are intrinsically dangerous and can react with macromolecules necessary for cell survival as well as adaptation. Mechanisms restricting access of otherwise cytotoxic ROS to targets important for their signaling function have been described and include their generally short half-lives, the various ROS-scavenging enzymes, the proximity of reactive target molecules, etc. But the Schumacker group’s findings that ROS produced from complex III in the mitochondria—a motile organelle—are directed away from the mitochondrial matrix and into the cytosol point to new concepts by which ROS compartmentalization could be engendered.

Abstract 75: Electron transport chain-dependent oxygen consumption in metastatic breast cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Although mitochondrial ATP-production deficiency has been historically implicated to account for the Warburg effect or “aerobic” glycolysis in cancer cells, recent observations indicate the presence of functionally competent mitochondria in cancer cells. We hypothesized that a lung-metastatic breast cancer cell line with intact mitochondrial membrane potential would exhibit mitochondria-dependent oxygen consumption. A murine breast cancer cell line (4T1) was used to evaluate mitochondrial membrane potential by JC-1 fluorescence microscopy. Oxygen consumption by cancer cells in suspension was determined using a dissolved oxygen meter. Fluorescence microscopy of JC-1 red aggregates indicated the presence of highly polarized mitochondria in 4T1 cells, suggesting that these cells are capable of oxidative phosphorylation. Oxygen consumption rate of 4T1 cells suspended in 10 mM glucose-containing medium equilibrated with 21% oxygen (258 µM dissolved oxygen) at 25°C was 2.98 nmol/min/million cells (Control). Oxygen consumption was significantly enhanced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler (7.88 nmol/min/million cells, p<0.05 vs. Control). Myxothiazol (a respiratory chain complex III inhibitor) inhibited the oxygen consumption (0.75 nmol/min/million cells; p<0.05 vs. Control), indicating the presence of a functional electron transport chain. A nitric oxide donor also inhibited oxygen consumption (0.84 nmol/min/million cells; p<0.05 vs. Control). These data indicate the presence of oxidative phosphorylation-competent mitochondria in breast cancer cells. We conclude that cancer cells possess respiration-capable mitochondria and their “aerobic” glycolysis may not reflect a compensatory mechanism for ATP generation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 75.